Inbred maize line R412H

ABSTRACT

The present invention is drawn to a novel DNA construct comprising an expression cassette having a constitutive promoter which functions in plant cells operably linked to a maize alcohol dehydrogenase intron, a DNA sequence of a gene encoding a Cry 1Ab protein, and a terminator functional in plants and optionally further comprising a second cassette including a promoter which functions in plants operably linked to a maize alcohol dehydrogenase intron, a DNA sequence of a gene encoding for phosphinothricin acetyl transferase, and a terminator functional in plants wherein the two cassettes are transcribed in the same direction. Also provided are transgenic plants, particularly maize plants, having such a construct stably incorporated into their genomes.

This application is a continuation of U.S. application Ser. No.09/042,426, filed Mar. 13, 1998, now U.S. Pat. No. 6,114,608, and acontinuation of Ser. No. 08/818,573, filed Mar. 14, 1997, the contentsof which are incorporated herein by reference, which claims the benefitsof U.S. application Ser. No. 60/109,808, filed Mar. 14, 1997, initiallyfiled as a regular U.S. application and subsequently converted to aprovisional U.S. application, the contents of which are incorporatedherein by reference.

BACKGROUND OF THE INVENTION

This invention relates to a novel promoter, a novel DNA constructcontaining the promoter and a Bt gene, and plants, especially cornplants, containing the novel DNA construct.

Bacillus thuringiensii (Bt) belongs to a large group of gram-positive,aerobic, endospore forming bacteria. During sporulation, these specificbacteria produce a parasporal inclusion body which is composed ofinsecticidally active crystalline protoxins, also referred to asδ-endotoxins.

These endotoxins are responsible for the toxicity of Bacillusthuringiensis to insects. The endotoxins of the various Bacillusthiunngiensis strains are characterized by high specificity with respectto target organisms. With the introduction of genetic engineering it hasbecome possible to create recombinant Bt strains which may contain achosen array of insect toxin genes, thereby enhancing the degree ofinsecticidal activity against a particular insect pest.

The insecticidal crystal proteins from Bt have been classified basedupon their spectrum of activity and sequence similarity (Hofte andWhiteley, Microbiol. Rev., 1989, 53:242-255 and Yamamoto and Powell,Advanced Engineered Pesticides, 1993, 3-42). Hofte and Whiteleypublished a classification scheme for the cry genes. Type I genes wereconsidered active only against Lepidoptera species; Type II genes wereactive against Lepidoptera and Diptera species; Type III genes wereactive against Coleoptera species and Type IV genes included both 70-and 130-kDa crystal protein and were highly active against mosquito andblackfly larvae. However, since this original classification many novelcry genes have been cloned and sequenced demonstrating that the originalsystem based on insect specificity required modification. Aclassification based on sequence homology along with new nomenclaturebased solely on amino acid identity has been proposed. (See Crickmore etal., Abstracts 28th Ann. Meeting Soc. Invert. Path. (1995), p14, Soc.Invert. Path., Bethesda Md.).

In this invention, the Cry proteins which are particularly effectiveagainst Lepidoptera species are preferred. These proteins are encoded bythe following nonlimiting group of genes: cry1Aa, cry1Ab, cry1Ac, cry1B,cry1C, cry1D, cry1E, cry1F, cry1G, cry2A, cry9C, cry5 and fusionproteins thereof. Among the cry genes, cry1Aa, cry1Ab, and cry1Ac showmore than 80% amino acid identity and cry1Ab appears to be one of themost widely distributed cry genes. The Cry1Ab proteins are particularlyeffective against larvae of Lepidoptera (moths and butterflies).

The ingestion of these proteins, and in some cases the spores, by thetarget insect is a prerequisite for insecticidal activity. The proteinsare solubilized in the alkaline conditions of the insect gut andproteolytically cleaved to form core fragments which are toxic to theinsect. The core fragment specifically damages the cells of the midgutlining, affecting the osmotic balance. The cells swell and lyse, leadingto eventual death of the insect.

A specific Lepidoptera insect, Ostrinia nubilalis (European corn borer(ECB)), causes significant yearly decrease in corn yield in NorthAmerica. One study reveales that approximately 10% of the corn acresplanted in the State of Illinois experienced a 9 to 15 percent annualyield loss, attributable solely to damage caused by the secondgeneration of corn borer. Other important lepidopteran insect pests ofcorn include Diatraea grandioselia (Southwestern Corn Borer),Helicoverpa zea (Corn Earworm) and Spodoptera frugiperda (FallArmyworm). The management practices of planting resistant or tolerantcorn hybrids and treatment with chemical and microbial insecticides havenot been satisfactory due to the low level of control provided byinsecticidal treatments and the lack of hybrid lines resistant to secondgeneration corn borers. Further tolerant and resistant hybrids often donot yield as well when infestation of ECBs are heavy. The use of corngenetically engineered to be resistant to specific corn insect pests hasmany advantages and these include a potential for substantial reductionin chemical insecticides and selective activity of the engineeredendotoxin which will not disrupt the population of beneficial non-targetinsect and animals.

Toxic Bt genes from several subspecies of Bt have been cloned andrecombinant clones have been found to be toxic to lepidopteran, dipteranand coleopteran insect larvae. However, in general, the expression offull length lepidopteran specific Bt genes has been less thansatisfactory in transgenic plants (Vaeck et al, 1987 and Barton et al,1987). It has been reported that the truncated gene from Bt kurstaki maylead to a higher frequency of insecticidal control. (U.S. Pat. No.5,500,365). Modification of the existing coding sequence by inclusion ofplant preferred codons including removal of ATTTA sequences andpolyadenylation signals has increase expression of the toxin proteins inplants. (U.S. Pat. No. 5,500,365). In the present invention a truncatedBt kurstaki HD-1 gene has been used.

The instant invention additionally includes a second coding segment. Thesecond coding segment comprises a DNA sequence encoding a selectivemarker for example, antibiotic or herbicide resistance including cat(chloramphenicol acetyl transferase), npt II (neomycinphosphototransferase II), PAT (phosphinothricin acetyltransferase), ALS(acetolactate synthetase), EPSPS (5-enolpyruvyl-shikimate-3-phosphatesynthase), and bxn bromoxynil-specific nitrilase). A preferred markersequence is a DNA sequence encoding a selective marker for herbicideresistance and most particularly a protein having enzymatic activitycapable of inactivating or neutralizing herbicidal inhibitors ofglutamine synthetase. The non-selective herbicide known as glufosinate(BASTA® or LIBERTY®) is an inhibitor of the enzyme glutamine synthetase.It has been found that naturally occurring genes or synthetic genes canencode the enzyme phosphinothricin acetyl transferase (PAT) responsiblefor the inactivation of the herbicide. Such genes have been isolatedfrom Streptomyces. These genes including those that have been isolatedor synthesized are also frequently referred to as bar genes. As usedherein the terms “bar gene” and “pat gene” are used interchangeably.These genes have been cloned and modified for transformation andexpression in plants (EPA 469 273 and U.S. Pat. No. 5,561,236). Throughthe incorporation of the pat gene, corn plants and their offspring canbecome resistant against phosphinothricin (glufosinate).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents a plasmid map of pZO960 which contains the Bt kurstakiexpression cassette.

FIG. 2 represents a plasmid map of the base transformation vector pZO997

FIG. 3 represents a plasmid map of pZO1500 which contains the PATcassette.

FIG. 4 represents a plasmid map of the (expression/transformation)vector pZO1502 which contains the Bt kurstaki cassette and the PATcassette.

SUMMARY OF THE INVENTION

The present invention is drawn to a novel recombinant DNA constructcomprising an expression cassette includes a constitutive promoter whichfunctions in plant cells operably linked to an intron that functions inmonocots; a DNA sequence of a gene encoding an insecticidal Bacillusthuringiensis protein toxin; and a terminator functional in plants; andoptionally further comprises a second cassette which includes a promoterwhich functions in plant cells operably linked to an intron thatfunctions in monocots; a DNA sequence of a gene encoding forphosphinothricin acetyl transferase; and a terminator functional inplants, wherein the two cassettes are transcribed in the same direction.

Therefore a first aspect of the present invention is a DNA constructwhich expresses the crystal protein toxin of a Bt effective againstLepidopteran insects at relatively high levels and further providesresistance to the non-selective herbicide glufosinate.

A second aspect of the invention is a plant transformation vectorcomprising the DNA construct as given above.

A third aspect of the present invention comprises a transformed plantcell including the DNA construct as given above wherein the DNA isstably incorporated in the plant genome.

A fourth aspect of the invention is a plant comprising transformed plantcells wherein the DNA construct as given above is stably incorporatedinto the genome of the plant.

The invention further encompasses plant seeds having the DNA constructas given above stably incorporated therein.

Another aspect of the invention includes a plant cell co-transformedwith a first nucleic acid construct comprising, a CaMV 35S constitutivepromoter which functions in plant cells operably linked to a maizealcohol dehydrogenase intron, a DNA sequence of a gene encoding a Cry1Abprotein toxin or a functionally related protein toxin, and a terminatorfunctional in plants and a second nucleic acid construct comprising aCaMV 35S promoter which functions in plant cells operably linked to amaize alcohol dehydrogenase intron, a DNA sequence of a gene encodingfor phosphinothricin acetyl transferase, and a terminator functional inplants wherein the first and second constructs are stably integrated inthe plant genome.

The DNA construct of the invention preferably is an expression cassettefunctional in a plant comprising a promoter functional in plants, forexample a CaMV 35S promoter, e.g., as disclosed in SEQ ID No. 1 or 5,preferably SEQ ID No. 1, operably linked to an intron which functions inmonocots, for example a maize alcohol dehydrogenase intron, e.g., asdisclosed in SEQ ID No. 2 or 6, preferably SEQ ID No. 2. Thispromoter/intron sequence is operably linked to a DNA sequence ofinterest, for example a gene encoding a Bt delta-δ-endotoxin, e.g.,encoding the toxin domain of a Cry1Ab protein or a functionally relatedtoxin protein, preferably modified for expression in plants, for exampleas depicted in SEQ ID No. 3, or a gene for a selectable marker, forexample a gene for herbicide resistance, preferably glufosinateresistance, for example a Pat gene, e.g., as depicted in SEQ ID No. 7.The gene of interest is suitably linked to a terminator functional inplants, e.g. a Nos terminator, for example as disclosed in SEQ ID No. 4or 8, preferably SEQ ID No. 4, to form an expression cassette functionalin a plant. Especially preferred embodiments of the Bt expressioncassette comprise SEQ ID Nos. 1, 2, 3 and 4 in operable sequence, e.g.,as in the Btk cassette described below. Especially preferred embodimentsof a Pat expression cassette comprise SEQ ID Nos. 5, 6, 7, and 8 inoperable sequence. In an especially preferred embodiment, a Btexpression cassette as described herein is linked on the same DNA with aPat expression cassette as described herein, e.g., a plasmid comprisingcassettes formed by SEQ ID Nos. 1-4 and 5-8 wherein the two cassettesare transcribed in the same direction, e.g., as in plasmid pZO1502.

The use of such expression cassettes in a method of transforming plants,e.g., maize plants, for example a method or biolistic or protoplasttransformation of maize plants, especially protoplast transformation asdescribed in the examples herein is also provided, as are plants stablytransformed with expression cassettes as described, particularly maizeplants, e.g., field corn, sweet corn, white corn, silage corn andpopcorn, and seed thereof. Particularly preferred are maize plants andseed thereof descended from the Bt11 transformation event described inExample 2, for example

Maize containing the Btk construct described within a 15 cM region ofchromosome 8, near position 117, in the approximate position of publicprobe UMC30a, in the interval flanked by two markers: Z1B3 and UMC150a,preferably

(i) elite inbred sweet corn lines R327H, R372H, R412H, R583H and R660H,

(ii) elite inbred field corn lines 2043Bt, 2044Bt, 2070Bt, 2100Bt,2114Bt, 2123Bt, 2227Bt, 2184Bt, 2124Bt, and 2221Bt, and

(iii) maize inbred varieties descended from the same transgenic event asthese lines which contain and express the same transgenic construct,

including seed thereof.

When particular inbred varieties are identified herein, it is understoodthat the named varieties include varieties which have the same genotypicand phenotypic characteristics as the identified varieties, i.e., arederived from a common inbred source, even if differently named. Theinvention also provides hybrid maize seed produced by crossing plants ofan inbred corn line as described above with plants of having a differentgenotype, and hybrid corn plants produced by growing such hybrid maizeseed. Also provided is a method of producing hybrid maize seedscomprising the following steps:

A. planting in pollinating proximity seeds of a first inbred maize lineas described herein and seeds of a second inbred line having a differentgenotype;

B. cultivating maize plants resulting from said planting until time offlowering;

C. emasculating said flowers of plants of one of the maize inbred lines;

D. allowing pollination of the other inbred line to occur, and

E. harvesting the hybrid seeds produced thereby.

Also provided are hybrid seeds produced by this method, F1 hybrid plantsproduced by growing such seeds, and parts of such F1 hybrid plants,including seeds thereof.

Seeds of the plants described herein (e.g., of maize plants, e.g., Bt11maize plants, for example inbred or hybrid seeds as described above) forplanting purposes is preferably containerized, e.g., placed in a bag orother container for ease of handling and transport and is preferablycoated, e.g., with protective agents, e.g., safening or pesticidalagents, in particular antifungal agents and/or insecticidal agents. Oneparticular embodiment of this invention is isolated inbred seed of theplants described herein, e.g. substantially free from hybrid seed orseed of other inbred seed, e.g., a seed lot or unit of inbred seed whichis at least 95% homogeneous, e.g., isolated seed of any of the maizeinbreds described in example 8 or 9 hereof.

Also provided herein, for the first time, are Bt maize varieties otherthan Bt field corn, particularly Bt sweet corn. Although Bt field cornhas been disclosed, it was not previously determined experimentallywhether or how a Bt delta δ-endotoxin would interact with traitsassociated with sweet corn, which is harvested at an earlier maturity(before it is dry), for a different purpose (usually fresh produce,canning or freezing, for human consumption) and has been bred thereforeto be qualitatively and quantitatively different from field corn in anumber of respects. Therefore, in one embodiment, the inventioncomprises a sweet corn comprising in its genome an expression cassettecomprising a coding region for a Bt delta-δ-endotoxin or functionalfragment or derivative thereof, under control of a promoter operable inmaize, e.g., an expression cassette as described herein. The sweet cornof the invention includes sweet or supersweet maize having a highersugar to starch ratio than field corn (e.g., yellow dent corn) due to areduced capacity to convert sugar into starch, typically characterizedby a sugary (su, e.g., su1) allele in the case of sweet corn, and/orshrunken allele (sh, e.g., sh2) or brittle allele (bt, e.g., bt2, not tobe confused with the gene for an endoxin from Bacillus thuringiensis,described elsewhere herein) in the case of supersweet corn, especiallymaize containing the su1 or sh2 alleles.

Bt maize of the invention, e.g., Bt11 maize, is found to be particularlysuited for the preparation of food materials (e.g., for human or animalconsumption, for example sweet corn for for packaging or fresh use as ahuman food, or grain or silage made from field corn) containing reducedlevels of fungal toxins, e.g., aflatoxins. While the mechanism is notentirely understood, in grain and silage made from Bt11 maize, the levelof aflatoxin is believed to be lower, possibly because the reduction ininsect damage reduces the level of opportunistic fungal infection in thegrowing plant. Accordingly, food materials made from Bt maize of theinvention, particularly Bt11 maize, for example grain and silage havingreduced levels of fungal toxins, particularly aflatoxins, and the use ofthe Bt maize of the invention in a method of preparing a foodstuff,especially grain or silage, with reduced levels of fungal toxins, e.g.,aflatoxins, is also provided.

DETAILED DESCRIPTION OF THE INVENTION

A promoter is defined as a nucleotide sequence at the 5′ end of astructural gene which directs the initiation of transcription. Thestructural gene is placed under regulatory control of the promoter.Various promoters which are active in plant cells are known anddescribed in the art. These include Cauliflower Mosaic Virus (CaMV) 19Sand 35S; nopaline synthase (NOS); mannopine synthase (MAS); actin;ubiquitin; ZRP; chlorophyll AB binding protein (CAB); ribulosebisphosphate carboxylase (RUBISCO); heat shock Brassica promoter (HSP80); and octopine synthase (OSC). The particular promoter used in thepresent invention should be capable of causing sufficient expression toresult in production of an effective amount of protein. The promoterused in the invention may be modified to affect control characteristicsand further may be a composite of segments derived from more than onesource, naturally occurring or synthetic. The preferred promoters areCaMV promoters and particularly CaMV 35S. The term “CaMV 35S” includesvariations of the promoter wherein the promoter may be truncated oraltered to include enhancer sequences, to increase gene expressionlevel, and composite or chimeric promoters, wherein portions of anotherpromoter may be ligated onto the CaMV 35S. A preferred embodimentincludes the 5′ untranslated region of the native 35S transcript, andmore particularly wherein the untranslated region includes about 100 to150 nucleotides. Additionally while 35S promoters are fairly homologous,any 35 S promoter in a preferred embodiment would include theuntranslated region of the native 35S transcript. Particularly preferred35S promoters are described in SEQ ID NO. 1 and SEQ ID NO. 5. Thepromoter as described in SEQ ID NO. 1 as part of the claimed constructmay have particular advantage in that the construct may be expressed inpollen tissue.

An intron is a transcribed nucleotide sequence that is removed from theRNA transcript in the nucleus and is not found in the mature mRNA. Suchsequences are well known in the art, and monocot introns include but arenot limited to sucrose synthetase (SS); glutathione transferase; actin;and maize alcohol dehydrogenase introns. An exon is part of a gene thatis transcribed into a mRNA and includes non-coding leader and/or trailersequences. An exon may code for a specific domain of a protein. Havingnative exon sequences around an intron may improve the introns splicingactivity or the ability of the nuclear splicesomal system to properlyrecognize and remove the intron. According to the invention, a preferredembodiment includes the native exon in the first cassette and moreparticularly 50 or more nucleotide bases of the native exon on each sideof the intron is preferred.

A gene refers to the entire DNA sequence involved in the synthesis of aprotein. The gene includes not only the structural or coding portion ofthe sequence but also contains a promoter region, the 3′ end and poly(A)sequences, introns and associated enhancers or regulatory sequences.

A structural heterologous gene is that part of a DNA segment whichencodes a protein, polypeptide or a portion thereof, and one which isnot normally found in the cell or in the cellular location where it isintroduced. The DNA sequence of a structural heterologous gene of thepresent invention include any DNA sequence encoding a crystal toxininsecticidal protein. The preferred toxins include but are not limitedto Cry1Aa, Cry1Ab, Cry1Ac, Cry1B, Cry1C, Cry1D, Cry1E, Cry1F, Cry1G,Cry2A, Cry2B, Cry3A, Cry3B, Cry3C, Cry4A, Cry4B, Cry4C, Cry4D, Cry5A,Cry9C, CytA and any fusion protein or truncated gene that encodes one ormore of the abovementioned toxins or a mixture thereof. Particularlypreferred toxins include Cry1Aa, Cry1Ab, Cry1Ac, Cry1C, Cry2A, Cry3C,Cry1E, Cry5A, Cry9C and any mixture or fusion protein thereof. In thepresent specification, the term fusion protein is used interchangeablywith the terms fusion toxin and hybrid protein and is a proteinconsisting of all or part of an amino acid sequence (known as a domain)of two or more proteins, and is formed by fusing the protein encodinggenes. An example of a DNA sequence useful in the cassette of thisinvention is a DNA sequence encoding a fusion toxin wherein the toxin isCry1Ab/Cry1C and Cry1E/Cry1C. The domains comprising the fusion proteinmay be derived from either naturally occurring or synthetic sources.

Many cry1Ab genes have been cloned and their nucleotide sequencesdetermined. A holotype gene sequence of cry1Ab has accession numberM13898 (The GenBank v. 70/EMBL v.29). A number of studies reveal thatthe amino terminal end of the Cry1A protein is responsible for theinsecticidal activity. This region depends on the particular protein butin general include a truncated gene that encodes from about amino acid25 to amino acid 610 of the protein.

In the present invention, a preferred cry1Ab gene includes a syntheticgene encoding the toxin domain of the protein produced by the Btkurstaki (k) HD-1 gene wherein the G+C content of the Btk gene isincreased and the polyadenylation sites and ATTTA regions are decreased.U.S. Pat. No. 5,500,365, which is hereby incorporated in its entiretydiscloses a synthetic Btk HD-1 and HD-73 gene, and truncated HD-1 andHD-73 genes. A particularly preferred cry1Ab gene of this invention isthe sequence as described in SEQ ID NO. 3.

Other preferred genes include those that are functionally equivalent tocry1Ab. These genes include all cry1Ab, cry1Aa, cry1Ac and variantsthereof wherein the expressed protein toxin is active against one ormore major maize Lepidoptera insect pests. The insect pests include theaforementioned European corn borer, Southwestern corn borer, Fallarmyworm, and Corn earworm.

The second structural gene that is part of the invention includes a DNAsequence encoding a selective marker for example, antibiotic orherbicide resistance including cat (chloramphenicol acetyl transferase),npt II (neomycin phosphototransferase II), PAT (phosphinothricinacetyltransferase), ALS (acetolactate synthetase), EPSPS(5-enolpyruvylshikimate-3-phosphate synthase), and bxn(bromoxynil-specific nitrilase). A preferred marker sequence is a DNAsequence encoding a selective marker for herbicide resistance and mostparticularly a protein having enzymatic activity capable of inactivatingor neutralizing herbicidal inhibitors of glutamine synthetase. Thenon-selective herbicide known as glufosinate (BASTA® or LIBERTY®) is aninhibitor of the enzyme glutamine synthetase. It has been found thatnaturally occurring genes or synthetic genes can encode the enzymephosphinothricin acetyl transferase (PAT) responsible for theinactivation of the herbicide. Such genes have been isolated fromStreptomyces. Specific species include Streptomyces hygroscopicus(Thompson C. J. et al., EMBO J., vol. 6:2519-2523 (1987)), Streptomycescoelicolor (Bedford et al, Gene 104: 39-45 (1991)) and Streptomycesviridochromogenes (Wohlleben et al. Gene 80:25-57 (1988)). These genesincluding those that have been isolated or synthesized are alsofrequently referred to as bar genes. As used herein the terms “bar gene”and “pat gene” are used interchangeably. These genes have been clonedand modified for transformation and expression in plants (EPA 469 273and U.S. Pat. No. 5,561,236). Through the incorporation of the pat gene,corn plants and their offspring can become resistant againstphosphinothricin (glufosinate). A preferred coding segment of a bar geneof the present invention is the sequence described in SEQ ID NO. 7.

The structural gene of this invention may include one or moremodifications in either the coding region or in the untranslated regionwhich would not substantially effect the biological activity or thechemical structure of the expression product, the rate of expression orthe manner of expression. These modifications include but are notlimited to insertions, deletions, and substitutions of one or morenucleotides, and mutations. The term homology as used herein refers toidentity or near identity of nucleotide or amino acid sequences. Theextent of homology is often measured in terms of percentage of identitybetween the sequences being compared. It is understood in the art thatmodification can occur in genes and that nucleotide mismatches and minornucleotide modifications can be tolerated and considered insignificantif the changes do not alter functionality of the final product. As inwell known in the art the various cry1A genes have very similar identityand reference in made to the article by Yamamoto and Powell, AdvancedEngineered Pesticides, 1993, 3-42 which includes a dendrogram tableshowing sequence homology among full length crystal proteins obtainedfrom the GenBank data base for a full length comparison.

Termination sequences are sequences at the end of a transcription unitthat signals termination of transcription. Terminators are 3′non-translated DNA sequences that contain a polyadenylated signal.Examples of terminators are known and described in the literature. Theseinclude but are not limited to nopline synthase terminator (NOS); the35S terminator of CaMV and the zein terminator.

Other elements may be introduced into the construct for examples matrixattachments region elements (MAR). These elements can be positionedaround an expressible gene of interest to effect an increase in overallexpression of the gene and to diminish position dependent effects uponincorporation into the plant genome.

Transformation means the stable integration of a DNA segment carryingthe structural heterologous gene into the genome of a plant that did notpreviously contain that gene. Co-transformation is transformation withtwo or more DNA molecules. Frequently one segment contains a selectablegene generally one for antibiotic or herbicide resistance.

As used herein the term plant tissue is used in a wide sense and refersto differentiated and undifferentiated plant tissue including but notlimited to, protoplasts, shoots, leaves, roots, pollen, seeds, callustissue, embryos, and plant cells (including those growing or solidifiedmedium or in suspension.

The DNA construct of this invention may be introduced into a planttissue by any number of art recognized ways. These included, but are notlimited to, direct transfer of DNA into whole cells, tissue orprotoplasts, optionally assisted by chemical or physical agents toincrease cell permeability to DNA, e.g. treatment with polyethyleneglycol, dextran sulfate, electroporation and ballistic implantation ofDNA coated particles. The following references further detail themethods available: Biolistic transformation or microprojectilebombardment (U.S. Pat. Nos. 4,945,050; 5,484,956; McCabe et al., AnnualRev. Genet. 22:421-477 (1988); Klein et al., Proc. Natl. Acad. Sci. USA,85:4305-4309 (1988); Klein et al., Bio/Technology 6:559-563 (1988);Gordon-Kamm et al., Plant Cell 2:603-618 (1990); and Vasil et al.,Bio/Technogy 11:1553-1558 (1993); Protoplast transformation—EPA 0 292435; EPA 0 465 875; and U.S. Pat. No. 5,350,689; microinjection—Crosswayet al., BioTechniques 4:320-334 (1986); direct gene transfer—Paszkoskiet al., EMBO J. 3:2717-2722 (1984); electrotransformation—U.S. Pat. No.5,371,003; and electroporation—Rigg et al., Proc. Natl, Acad. Sci. USA83: 5602-5606 (1986). Transformation is also mediated by Agrobacteriumstrains, notably A. tumefaciens and A. rhizogenes, and also by variousgenetically engineered transformation plasmids which include portions ofthe T-DNA of the tumor inducing plasmids of Agrobacteria. EPA 0 604662A1, Japan Tobacco Inc.; Hinchee et al., BioTechnology 6: 915-921(1988). Also see Potrykus, I. Annu. Rev. Plant Physiol. Plant Mol. Biol.1991, 42:205-225. The choice of a particular method may depend on thetype of plant targeted for transformation.

Transformed plants may be any plant and particularly corn, wheat,barley, sorghum, and rice plants, and more particularly corn plantsderived from a transformant or backcrossing through further breedingexperiments.

EXAMPLE 1

Plasmid construction:

A. Plasmid pZO1502 construction: The plasmid pZO1502 can be consideredto consist of three basic regions; the base plasmid vector, anexpression cassette for the Btk gene, and an expression cassette for thepat gene. For convenience, the various parts were constructed separatelyand then combined into the final plasmid. In order to assemble thedesired elements for the Btk and pat gene expression cassettes, therestriction sites used to generate the desired elements sometimesrequired modification. The following example demonstrates the procedureused to produce the pZO1502 plasmid. One skilled in the art could devisealternate ways to construct the final transformation plasmid.

B. Base Plasmid Vector: The base vector, pUC18 (GenBank accessionL08752, Norrander, J. M., et al., 1983. Gene 26:101-106), was modifiedby replacing the EcoO 109 I restriction site with a Bgl II linker(digestion with EcoO 109 1, fill in with T4 polymerase, and addition ofa Bgl II linker). This base vector was further modified to replace theBspH I sites at 1526 and 2534 with Not I restriction sites (vector cutwith BspH I, filled in, and replaced with Stu I linkers; the Stu I sitewas then cut and Not I linkers added). The addition of the Not Irestriction sites provided a convenient way to produce a linear DNAfragment containing the two desired gene cassettes (Btk and pat)separated from the ampicillin gene sequence (required for plasmidproduction in E. coli). This linearization also significantly increasedprotoplast transformation frequency. The final base vector was namedpZO997B (FIG. 2).

C: Btk gene expression cassette: The Dde I to Dde I fragment of the 35Spromoter from cauliflower mosaic virus (strain CM1841, GenBank accession# V00140, Gardner, R. C., et al., 1981. Nucleic Acids Res. 9:2871-2888)(SEQ ID NO. 1) was converted to Sac I by addition of linkers and clonedinto the Sac I site of the polylinker region of a pUC19 based vector.The sixth intron from maize Adh1-1S gene (GenBank accession X04049,Dennis, E. S., et al., 1984. Nucleic Acid Res. 12:3983-4000) wasisolated as a Pst I to Hpa II fragment, converted with BamH I linkers(SEQ. ID NO. 2), and cloned into the BamH I poly linker site 3′ to the35S promoter. The 3′ terminator from Nopaline synthetase, NOS, (GenBankaccession V00087, Bevan, M., et al., 1983. Nucleic Acids Res.11:369-385) (SEQ. ID NO 4) was isolated as ˜250 bp fragment with Pst Iand Bgl H. The Bgl II site was polished with T4 polymerase, a Hind IIIlinker added, and the fragment inserted behind a gus gene constructusing the Pst I and Hind III sites. The gus gene was cloned into the SalI to Pst I site of the polylinker. The gus construct utilized asynthetic linker (Sal I to Nco I, which provides for an A nucleotide atthe −3 position from the translation start ATG: GTCGACCATGG) (SEQ ID NO.9). The Pst I site was then trimmed, a Bcl I linker added, and the gusgene sequence replaced with a synthetic gene encoding a cry1Ab toxin(SEQ. ID NO. 3) as a Nco I to Bgl II insert to produce the vector pZO960(FIG. 1)

D. Pat zene expression cassette: Although composed of similar elements,the pat expression cassette was derived from a different series ofcloning steps. The 35S promoter (SEQ ID NO. 5) was obtained as a Hinc IIto Dde I fragment from the cauliflower mosaic virus (strain CABB-S,GenBank accession # V00141, Franck, A., et al., 1980. Cell 21: 285-294)and converted to BamH I—Xba I with linkers. The second intron sequencefrom maize Adh1-1S (GenBank accession X04049, Dennis, E. S., et al.,1984. Nucleic Acid Res. 12:3983-4000) (SEQ ID NO. 6) was isolated as aXho II to Xho II fragment and cloned into the BamH I site of pUC12,converting the Xho II sites to BamH I. As a BamH I fragment it wascloned into the Bgl II site of a synthetic polylinker (Asu II, Bgl II,and Xho I). The Asu II site was then filled in and ligated to the(filled in) Xba I site of the 35S promoter fragment. The synthetic patgene sequence was subcloned from plasmid pOAC/Ac (obtained from Dr.Peter Eckes, Massachusetts General Hospital, Boston Mass.) (SEQ ID NO.7) as a Sal I to Pst I fragment and combined with the 35S/Adhivs2promoter (Xho I) and the 3′ NOS terminator sequence Pst I to Bgl II(GenBank accession V00087, Bevan, M., et al., 1983. Nucleic Acids Res.11:369-385) (SEQ ID NO. 8). These pieces were all combined with thepZO997B base vector to produce the pat expression vector pZO1500 (FIG.3).

As the final construction step, the Btk expression cassette wassubcloned from pZO960 as an EcoR I-Hind III fragment and inserted intothe EcoR I-Hind m polylinker site of pZO1500 to produce the finalvector, pZO1502 (FIG. 4). The amp (beta-lactamase) gene was removedprior to plant transformation by digestion with NotI. pZO1502 has beendeposited with the American Type Culture Collection (ATCC), 12301Parklawn Drive, Rockville, Md. 20852-1776 USA pursuant to the BudapestTreaty prior to the filing of this application and accorded accessionnumber 209682, and the complete sequence of this plasmid is disclosed inSEQ. ID No. 9.

EXAMPLE 2

Protoplast transformation, selection of transformed corn cells andregeneration:

The initial parental transformation of the corn line to be planted wasaccomplished through insertion of a DNA fragment from plasmid pZO1502,containing the two cassettes of Btk and the pat gene, into the genome ofa proprietary corn cell line owned by Hoerchst AG (Frankfurt Germany).The transformation was performed using a protoplast transformation andregeneration system as described in detail in European PatentApplication Publication Number 0 465 875 A, published Jan. 15, 1992 andEuropean Patent Application Publication Number 0 469 273 A, publishedFeb. 5, 1992 and Theor. Appl. Gent. 80:721-726 (1990)). The contents ofwhich are hereby incorporated by reference.

After some weeks on selective media putative transformant clumps ofcells were observed and transformed protoplasts were selected in vitrowith a glufosinate-ammonium herbicide. Sixteen leaf producinggenetically transformed corn lines were obtained from protoplaststreated with the gene expression cassette from pZO1502. One of theselines was designated as transformant number 11. This transformant wasgrown to maturity.

The Bt-11 R0 transformed plants were pollinated with nontransformedNorthrup King elite inbred male parents and R₁ seed was collected.Descendants of the initial crossing have been successively backcrossedand test crossed to establish and evaluate corn lines carrying the Btkgene. Such lines are described more fully in the Examples 8 and 9 belowand have been deposited with the ATCC pursuant to the Budapest Treaty.

EXAMPLE 3

Stable Transformation:

Expression of the Btk gene was tested by transforming the Bt gene vectorpZO960 into BMS (Black Mexican Sweet) corn cells. Protoplasts wereisolated from a suspension culture BMS cell line and electroporated toinduce DNA uptake essentially as described in Sinibaldi, R. M. andMettler, I. J., 1992, In: Progress in Nucleic Acid Research andMolecular Biology (W. E. Cohn and K. Moldave, eds.) Academic Press, SanDiego, vol. 42:229-259. Cells which had stably incorporated DNA wereselected by co-transformation with a plasmid containing a kanamycinresistance selectable gene. A number of independent transgenic eventswere selected by the expression of the antibiotic resistance tokanamycin. Approximately 1 gram of each transgenic line was then used totest for biological activity against neonate larvae of Manducca sexta.Control, non-transformed, BMS callus tissue supported normal growth ofthe larvae throughout the test period. Transgenic callus lines were thenrated for the degree of growth inhibition. As shown in Table 1, out of33 BMS lines co-transformed with pZO960, 6 lines were positive forinsecticidal activity showing complete growth inhibition and 100%mortality within 2 or three days. Quantitative Elisa assays showed thatthe transgenic tissues produced an average of 3.1 ng of Bt protein permg of total extracted protein.

TABLE 1 Stable Transformation with Btk Cassette Insect activity Bt ELISAassays Construct #pos/#test ng/mg protein pZO960 6⁺/33 3.1 ⁺ = stronginsecticidal activity, 100% mortality in 2-3 days, little feeding.

EXAMPLE 4

Insertion Site of Bt11 Transgenic Event:

The original genetic stock into which the Btk sequence was transformedwas designated HE89. The Ro plants were used as the female parent forinitial crosses to two, elite Northrup King proprietary inbred lines forwhich Btk-conversion was sought. Multiple backcrosses were conductedinto many additional inbred lines with individuals selected thatcontained the insertion sequence but were, otherwise, as similar to theelite recurrent parents as possible. Four or more backcrosses andselfing to homozygosity were used in the conversion process. Finishedconversion stocks were evaluated with a series of 50 or 60 RFLP probesselected to be well distributed throughout the genome. Genotypes of theBtk converted inbreds were compared to those of their recurrent parentisolines. They were generally identical or nearly identical for allgenetic markers, except for three probes on a small segment of the longarm of chromosome 8. All conversion stocks differ from the genotype ofthe transformed stock, HE89, for this segment, thus differing from therecurrent parents. There were no other genomic regions with consistentdifferences between Btk-conversions and their recurrent parents. Thesethree probes exist within 10 centiMorgans(cM) of one another at theapproximate position of the public probe UMC30a, which has been placedat map position 117 in the 1995 map of RFLP probe positions distributedby the University of Missouri at Columbia.

A series of 95 backcross progeny were further characterized withnumerous probes in the region of chromosome 8 identified above. The sizeof the “donor” DNA segment varied among these progeny. However, five ofthe progeny failed to contain the donor alles at the flanking markers:Z1B3 and UMC150a, despite presence of the Btk sequence. These two probesare approximately 15 cM apart on chromosome 8. Thus, the insertion siteis within a 15 cM region on the long arm of chromosome 8, near position117, and in the interval flanked by two markers: Z1B3 and UMC150a

Southern Analysis of the Transgenic Event:

The Bt11 transgenic seeds backcrossed into inbred line HAF031 were sownin the greenhouse and sprayed with BASTA herbicide at the four leafstage. Resistant plants and control, untransformed, HAF031 inbred plantswere then used for DNA extraction and Southern blot analysis (T.Maniatis, E. F. Fritsch and J. Sambrook, 1982, Molecular Cloning ALaboratory Manual, Cold Spring Harbor Laboratory) The genomic DNAsamples were digested with following restriction enzymes and probed withlabeled DNA for Btk and PAT gene sequences. The first enzyme combinationutilized 2 restriction sites present on the plasmid DNA. The next twoenzymes had only one known location and would be expected to cut thegenomic DNA at a distant site in the plant DNA. The actual size of anyobserved fragment depends on the insertion event. The number of bandscan be used to estimate insertion copy number—each gene copy wouldproduce a unique band on the Southern blot.

The results of a Southern blot are summarized in Table 2 These data showthat the Bt11 transgenic lines are derived from a single insertion eventcontaining one gene copy of the Bt and pat gene sequences.

TABLE 2 Restriction Enzymes Probe Predicted - Observed #Fragment Sal 1and Sac I Btk 1.3 kb  1.3 kb 1 Hind III Btk  ≧3 kb ˜30 kb 1 EcoR I Btk ≧5 kb ˜25 kb 1 PstI and Hind III PAT 1.5 kb  1.5 kb 1 Hind III PAT  >2kb ˜30 kb 1 EcoR I PAT  >5 kb ˜25 kb 1

The DNA probe fragments were isolated from the original plasmid vectorpZO1502: Btk=Sal I and Sac I fragment and PAT+Sal I fragment.

EXAMPLE 5

Enzymatic Activity of PAT in the Bt Transformed Lines:

Fresh tissue samples (30-50 mg) were ground on ice in ˜5 volumes ofextraction buffer (100 mM Tris-HCL, pH 7.5), 3 mg/ml dithiothreitol and0.3 mg/ml bovine serum albumin (BAS fraction V). The homogenate wascentrifuged to clarity (12,000×g for 5 min). Approximately 2 μl ofextract was added to the reaction mixture containing the extractionbuffer plus 125 μM acetyl CoA and 250 μM phosphinothricin. The enzymaticreaction was allowed to proceed for 1 hour at 37° C. The reaction mixwas then spotted onto TLC silica gel plates (Baker Si250-PA (19C)). Theplate was chromatographed for 2-3 hours with isopropanol:NH4OH (3:2),air dried and vacuum dried in an oven at 80° C. The plates were thenexposed to X-ray film for 1-4 days. The results of a typical assayconfirm the presence and enzymatic activity of the PAT protein in the Btlines.

EXAMPLE 6

Inheritance and Gene Stability:

The segregation of the Btk gene and the PAT gene were followed inmultiple generations. Eight F1 corn plants identified as containing theBtk and PAT genes were selfed to produce a S1 population. The S1population was screened for resistance to ECB and Ignite® herbicide. Allplants were either resistant to ECB and Ignite or susceptible to both.The segregation ratios were consistent with an expected ratio of 3:1 fora single dominant locus.

EXAMPLE 7

Bt-11 maize versus European Corn Borer Field Trials:

Trials were conducted using a randomized complete block design. Tworeplicates were planted at three locations across three states intwo-row plots. Hybrids were grouped according to relative maturity andplanted at appropriate sites based on maturity. Southern trialscontained six Btk hybrids and four non-Btk control hybrids. The northerntrials consisted of eight Btk hybrids and two non-Btk hybrids. Plantswere artificially infected as they approached the V6 stage of growth.Approximately fifty larvae were applied to ten plants in the first rowof each plot every three to four days over a two and one-half weekperiod. By the end of the first generation infesting, each plant hadbeen infected with at least 200 neonate larvae. Just prior to tasselemeregnce, 1-9 leaf damage ratings were assigned to each of the tenplants per plot. The rating scale of Gurthie, W. D., et al. (1960, “Leafand Sheath Feeding Resistance to the European Corn Borer in Eight InbredLines of Dent Corn”, Ohio Ag. Exp, Sta. Res. Bull. 860) was used,wherein 1=no damage or few pinholes, 2=small holes on a few leaves,3=shot-holes on serval leaves, 4=irregular shaped holes on a few leaves,and 9=several leaves with many emerging elongated lesions.

As plants began to shed pollen, second generation ECB infestation began.The first ten plants of the first row of each plot were infected with40-50 larvae every three to four days over a two and one-half weekperiod. Eventually every plant had been infected with approximately 200more larvae. After approximately 45 to 50 days, plants were dissectedfrom top to the ground and the total length of tunnels created by ECBfeeding was estimated and converted to centimeters for reporting.Analysis of Variance and Least Significant Difference mean separationwere used to analyze the results.

Average leaf feeding damage scores were approximately 3.9 on non-Btkhybrids and 1.1 for Btk hybrids wherein 1 on the scale of 1 to 9represents no damage. Average stalk damage represented as centimeterstunneled per plant, was approximately 4.9 cm in the non-Btk controlhybrids. The Btk hybrids displayed only approximately 0.2 cm oftunneling per plant. In all cases, the difference between Btk hybridsand non-Btk hybrids was significant at a P-value of less than 0.01 basedon AVOVA and LSD mean separation. Field tests conducted to determinedthe resistance of Btk hybrids and non-Btk hybrids for Southwestern CornBorer and Fall Armyworm also indicated that Btk hybrids showed excellentpotential for assisting in the control of these insect pests.

EXAMPLE 8

Bt11 Sweet Corn

Inbred backcrossing of Bt11 event material as described in Example 4into Novartis (Rogers) elite inbred sweet corn lines was carried out toobtain Bt11 inbred sweet corn lines, including inbreds R327H, R372H,R412H, R583H and R660H. These inbreds and their F1 hybrid progeny allcontain the Btk insert as described above at the location describedabove and exhibit insect resistance and herbicide resistance as for theother lines descended from the Bt11 event. For example, 2500 seeds ofeach of these lines were deposited with ATCC prior to the filing of thisapplication pursuant to the Budapest Treaty and accorded accessionnumbers as follows: R327H: ATCC Accession No: 209673, deposited Mar. 11,1998, R372H: ATCC Accession No: 209674, deposited Mar. 11, 1998, R412H:ATCC Accession No: 209675, deposited Mar. 11, 1998, R583H: ATCCAccession No: 209671, deposited Mar. 11, 1998 and R660H: ATCC AccessionNo: 209672, deposited Mar. 11, 1998. These lines were evaluated atNampa, Idaho and Stanton, Minnesota during the summer and fall of 1997,and characterized in relation to a standard reference inbred (Iowa5125,from North Central Region Plant Introduction Center, Ames, Iowa) havingsimilar background and maturity, as depicted on the following table.(All measurements are in centimeters unless otherwise noted. Colors areaccording to Munsell color code chart.)

TABLE 3 Trait R327H R372H R412H R583H R660H Iowa5125 Kernel colorYellow- Yellow- Yellow- Yellow- Yellow- Yellow- orange orange orangeorange orange orange Endosperm type sul sul sul sh2 sh2 sul Maturity(days) emergence to 50% silk 71 70 75 70 77 71 emergence to 50% pollen68 67 68 66 73 67 50% silk to optimal edible 24 26 25 25 29 25 qualityPlant plant height 207.0 199.7 144.0 173.8 174.8 152.8 ear height 51.865.9 45.3 40.1 57.0 57.5 top ear internode 17.6 15.5 10.0 15.8 13.6 13.8avg. number of tillers 2.3 1.1 0.4 3.3 1.2 0.8 avg. number of ears/stalk1.8 1.9 1.7 2.1 2.0 1.3 anthocyanin of brace roots absent absent absentabsent absent absent Leaf width of ear node leaf 7.5 6.4 8.1 7.5 9.7 7.3length of ear node leaf 70.7 65.0 54.0 64.1 67.3 82.4 no. of leavesabove top ear 6 5 5 5 6 6 degrees of leaf angle 49 41 63 46 60 56 leafcolor very dark very dark green- very green- green- green green yellowdark yellow yellow green Tassel no. of primary lateral 15 9 16 10 16 28branches tassel length 45.8 42.0 31.0 41.6 34.5 28.4 Ear silk colorgreen- green- green- green- light light green yellow yellow yellowyellow green position at dry husk stage upright pendent horizontal —upright pendent ear length 14.5 16.0 15.3 16.7 15.7 13.3 ear diameter atmidpoint 4.1 3.8 3.74 4.67 4.05 5.33 number of kernel rows 16 16 16 1516 21 cob diameter at midpoint 2.59 2.50 2.53 2.61 2.54 2.94

EXAMPLE 9

Bt11 Field Corn

Inbred backcrossing of Bt11 event material as described in Example 4into Novartis (Rogers) elite inbred field corn lines was carried out toobtain Bt11 inbred field corn lines, for example Yellow Dent inbredlines 2044Bt, 2070Bt, 2100Bt, 2114Bt, 2123Bt, 2227Bt, 2184Bt, 2124Bt,and 2221Bt. These inbreds and their hybrid progeny all contain the Btkinsert as described above at the location described above and exhibitinsect resistance and herbicide resistance as for the other plantsdescended from the Bt11 event. 2500 seeds of each of the following lineswere deposited with ATCC pursuant to the Budapest Treaty and accordeddeposit numbers as follows: 2044Bt: ATCC 203943, 2070Bt: ATCC 203941,2227Bt: ATCC 203942, 2184Bt: ATCC 203944, and 2221Bt:. Bt11 inbreds werealso made by marker assisted inbred conversion of the following lines,NP948 (ATCC 209406), NP2017 (ATCC 209543), NP904 (ATCC 209458), NP2010(ATCC), all deposited with ATCC pursuant to the Budapest Treaty toobtain 2100Bt, 2114Bt, 2123Bt and 2124Bt respectively.

Hybrids from Bt11 inbred conversions were evaluated extensively againsthybrids from isogenic, non-transgenic parents in a number of fieldtrials. In general, there was a significant yield advantage to the BT11version. There was no attempt to control natural infestations ofEuropean Corn Borers in these trial locations. Grain moisture at harvestis sometimes slightly higher in the BT11 version. This can often beattributed to the improved plant health, due to reduced stalk rot. Insome cases, grain test weight is higher in the BT11 version, which canalso reduce the rate of grain dry down. Stalk lodging is typically lowerin the BT11 versions. Push test and Late season intactness are alsotypically better in BT11 versions. In some cases, stay green is better.Plant and ear height are sometimes slightly higher in the BT11 version.For other traits, no consistent detrimental changes in performance havebeen observed.

2124Bt, 2221Bt, and 2070Bt are southern (late) maturities, whereas2044Bt, 2100Bt, 2114Bt, 2227Bt, 2184Bt, and 2123Bt are northern (early)maturities. These inbred Bt lines have the following generalcharacterization:

2044Bt—dark-reddish purple silk, slight pale green color, very slightlyfaded chlorotic stripes in leaves, medium tall, medium ear placement,purple tip to glume

2100Bt—green-yellow silk, medium-short plant height, medium low earplacement, green with purple glume, light green overall appearance

2114Bt—dark reddish purple silk, small tassel, slight crook in stalknodes, slight pale green color, medium tall, medium ear placement,higher yielding than 2044Bt

2227Bt—very thin loose husk at harvest, root lodges, medium plantheight, medium ear placement

2184Bt—medium plant height, medium ear placement, very light pollenshedder, green yellow silk color, pale purple anther

2123Bt—green with purple glumes, purple anther, green yellow silk,medium plant height

Sequence information Sequence 1: 35S promoter (EcoR I, Sac I, -35S- SacI, Kpn I, Sma I)   1 AATTCGAGCT CGTCAGAAGA CCAGAGGGCT ATTGAGACTTTTCAACAAAG GGTAATATCG  61 GGAAACCTCC TCGGATTCCA TTGCCCAGCT ATCTGTCACTTCATCGAAAG GACAGTAGAA 121 AAGGAAGGTG GCTCCTACAA ATGCCATCAT TGCGATAAAGGAAAGGCTAT CGTTCAAGAT 181 GCCTCTACCG ACAGTGGTCC CAAAGATGGA CCCCCACCCACGAGGAACAT CGTGGAAAAA 241 GAAGACGTTC CAACCACGTC TTCAAAGCAA GTGGATTGATGTGATATCTC CACTGACGTA 301 AGGGATGACG CACAATCCCA CTATCCTTCG CAAGACCCTTCCTCTATATA AGGAAGTTCA 361 TTTCATTTGG AGAGGACACG CTGAAATCAC CAGTCTCTCTCTACAAATCT ATCTCTCTCT 421 ATTTTCTCCA TAATAATGTG TGAGTAGTTC CCAGATAAGGGAATTAGGGT TCTTATAGGG 481 TTTCGCTCAC GTGTTGAGCA TATAAGAAAC CCTTACGAGCTCGGTACCCG GG Sequence 2: Adh1-1S intron 6 (BanmHI, -ADH1SIVS6-, BamHi,Xba I, Sal I)   1 GATCCGGAAG GTGCAAGGAT TGCTCGAGCG TCAAGGATCA TTGGTGTCGACCTGAACCCC  61 AGCAGATTCG AAGAAGGTAC AGTACACACA CATGTATATA TGTATGATGTATCCCTTCGA 121 TCGAAGGCAT GCCTTGGTAT AATCACTGAG TAGTCATTTT ATTACTTTGTTTTGACAAGT 181 CAGTAGTTCA TCCATTTGTC CCATTTTTTC AGCTTGGAAG TTTGGTTGCACTGGCACTTG 241 GTCTAATAAC TGAGTAGTCA TTTTATTACG TTGTTTCGAC AAGTCAGTAGCTCATCCATC 301 TGTCCCATTT TTTCAGCTAG GAAGTTTGGT TGCACTGGCC TTGGACTAATAACTGATTAG 361 TCATTTTATT ACATTGTTTC GACAAGTCAG TAGCTCATCC ATCTGTCCCATTTTTCAGCT 421 AGGAAGTTCG GTTGCACTGA ATTTGTGAAC CCAAAAGACC ACAACAAGCCGCGGATCCTC 481 TAGAGTCGAC Sequence 3: cry1Ab toxic gene region (Nco I,-cry1Ab,-, Bgl II)    1 CATGGACAAC AACCCAAACA TCAACGAATG CATTCCATACAACTGCTTGA GTAACCCAGA   61 AGTTGAAGTA CTTGGTGGAG AACGCATTGA AACCGGTTACACTCCCATCG ACATCTCCTT  121 GTCCTTGACA CAGTTTCTGC TCAGCGAGTT CGTGCCAGGTGCTGGGTTCG TTCTCGGACT  181 AGTTGACATC ATCTGGGGTA TCTTTGGTCC ATCTCAATGGGATGCATTCC TGGTGCAAAT  241 TGAGCAGTTG ATCAACCAGA GGATCGAAGA GTTCGCCAGGAACCAGGCCA TCTCTAGGTT  301 GGAAGGATTG AGCAATCTCT ACCAAATCTA TGCAGAGAGCTTCAGAGAGT GGGAAGCCGA  361 TCCTACTAAC CCAGCTCTCC GCGAGGAAAT GCGTATTCAATTCAACGACA TGAACAGCGC  421 CTTGACCACA GCTATCCCAT TGTTCGCAGT CCAGAACTACCAAGTTCCTC TCTTGTCCGT  481 GTACGTTCAA GCAGCTAATC TTCACCTCAG CGTGCTTCGAGACGTTAGCG TGTTTGGGCA  541 AAGGTGGGGA TTCGATGCTG CAACCATCAA TAGCCGTTACAACGACCTTA CTAGGCTGAT  601 TGGAAACTAC ACCGACCACG CTGTTCGTTG GTACAACACTGGCTTGGAGC GTGTCTGGGG  661 TCCTGATTCT AGAGATTGGA TTAGATACAA CCAGTTCAGGAGAGAATTGA CCCTCACAGT  721 TTTGGACATT GTGTCTCTCT TCCCGAACTA TGACTCCAGAACCTACCCTA TCCGTACAGT  781 GTCCCAACTT ACCAGAGAAA TCTATACTAA CCCAGTTCTTGAGAACTTCG ACGGTAGCTT  841 CCGTGGTTCT GCCCAAGGTA TCGAAGGCTC CATCAGGAGCCCACACTTGA TGGACATCTT  901 GAACAGCATA ACTATCTACA CCGATGCTCA CAGAGGAGAGTATTACTGGT CTGGACACCA  961 GATCATGGCC TCTCCAGTTG GATTCAGCGG GCCCGAGTTTACCTTTCCTC TCTATGGAAC 1021 TATGGGAAAC GCCGCTCCAC AACAACGTAT CGTTGCTCAACTAGGTCAGG GTGTCTACAG 1081 AACCTTGTCT TCCACCTTGT ACAGAAGACC CTTCAATATCGGTATCAACA ACCAGCAACT 1141 TTCCGTTCTT GACGGAACAG AGTTCGCCTA TGGAACCTCTTCTAACTTGC CATCCGCTGT 1201 TTACAGAAAG AGCGGAACCG TTGATTCCTT GGACGAAATCCCACCACAGA ACAACAATGT 1261 GCCACCCAGG CAAGGATTCT CCCACAGGTT GAGCCACGTGTCCATGTTCC GTTCCGGATT 1321 CAGCAACAGT TCCGTGAGCA TCATCAGAGC TCCTATGTTCTCATGGATTC ATCGTAGTGC 1381 TGAGTTCAAC AATATCATTC CTTCCTCTCA AATCACCCAAATCCCATTGA CCAAGTCTAC 1441 TAACCTTGGA TCTGGAACTT CTGTCGTGAA AGGACCAGGCTTCACAGGAG GTGATATTCT 1501 TAGAAGAACT TCTCCTGGCC AGATTAGCAC CCTCAGAGTTAACATCACTG CACCACTTTC 1561 TCAAAGATAT CGTGTCAGGA TTCGTTACGC ATCTACCACAAACTTGCAAT TCCACACCTC 1621 CATCGACGGA AGGCCTATCA ATCAGGGTAA CTTCTCCGCAACCATGTCAA GCGGCAGCAA 1681 CTTGCAATCC GGCAGCTTCA GAACCGTCGG TTTCACTACTCCTTTCAACT TCTCTAACGG 1741 ATCAAGCGTT TTCACCCTTA GCGCTCATGT GTTCAATTCTGGCAATGAAG TGTACATTGA 1801 CCGTATTGAG TTTGTGCCTG CCGAAGTTAC CTTCGAGGCTGAGTACTAGC A Sequence 4: NOS terminator (Bcl I, -NOS-, Hind III)   1GATCAGGATC GTTCAAACAT TTGGCAATAA AGTTTCTTAA GATTGAATCC TGTTGCCGGT  61CTTGCGATGA TTATCATATA ATTTCTGTTG AATTACGTTA AGCATGTAAT AATTAACATG 121TAATGCATGA CGTTATTTAT GAGATGGGTT TTTATGATTA GAGTCCCGCA ATTATACATT 181TAATACGCGA TAGAAAACAA AATATAGCGC GCAACCTAGG ATAAATTATC GCGCGCGGTG 241TCATCTATGT TACTAGATCC A Sequence 5: 35S promoter (BamH I, -35S-, Xba I)  1 GATCCGAACA TGGTGGAGCA CGACACGCTT GTCTACTCCA AAAATATCAA AGATACAGTC 61 TCAGAAGACC AAAGGGCAAT TGAGACTTTT CAACAAAGGG TAATATCCGG AAACCTCCTC121 GGATTCCATT GCCCAGCTAT CTGTCACTTT ATTGTGAAGA TAGTGGAAAA GGAAGGTGGC181 TCCTACAAAT GCCATCATTG CGATAAAGGA AAGGCCATCG TTGAAGATGC CTCTGCCGAC241 AGTGGTCCCA AAGATGGACC CCCACCCACG AGGAGCATCG TGGAAAAAGA AGACGTTCCA301 ACCACGTCTT CAAAGCAAGT GGATTGATGT GATATCTCCA CTGACGTAAG GGATGACGCA361 CAATCCCACT ATCCTTCGCA AGACCCTTCC TCTATATAAG GAAGTTCATT TCATTTGGAG421 AGGACACGCT GAAATCACCA GTCTCTCTCT ACAAATCTAT CTCTCTCTAT AATAATGTGT481 GAGTAGTTCC CAGATAAGGG AATTAGGGTT CTTATAGGGT TTCGCTCATG TGTTGAGCAT541 ATAAGAAACC CTTACTCTAG Sequence 6: Adh1-1s intron 2 (partial Asu II-ADH1SIVS2-, Xho I)   1 CGAAGATCCT CTTCACCTCG CTCTGCCACA CCGACGTCTACTTCTGGGAG GCCAAGGTAT  61 CTAATCAGCC ATCCCATTTG TGATCTTTGT CAGTAGATATGATACAACAA CTCGCGGTTG 121 ACTTGCGCCT TCTTGGCGGC TTATCTGTCT CAGGGGCAGACTCCCGTGTT CCCTCGGATC Sequence 7: Pat gene (Sal I, -Pat-, Bgl II, Sal I.Pst I)   1 TCGACATGTC TCCGGAGAGG AGACCAGTTG AGATTAGGCC AGCTACAGCAGCTGATATGG  61 CCGCGGTTTG TGATATCGTT AACCATTACA TTGAGACGTC TACAGTGAACTTTAGGACAG 121 AGCCACAAAC ACCACAAGAG TGGATTGATG ATCTAGAGAG GTTGCAAGATAGATACCCTT 181 GGTTGGTTGC TGAGGTTGAG GGTGTTGTGG CTGGTATTGC TTACGCTGGGCCCTGGAAGG 241 CTAGGAACGC TTACGATTGG ACAGTTGAGA GTACTGTTTA CGTGTCACATAGGCATCAAA 301 GGTTGGGCCT AGGATCCACA TTGTACACAC ATTTGCTTAA GTCTATGGAGGCGCAAGGTT 361 TTAAGTCTGT GGTTGCTGTT ATAGGCCTTC CAAACGATCC ATCTGTTAGGTTGCATGAGG 421 CTTTGGGATA CACAGCCCGG GGTACATTGC GCGCAGCTGG ATACAAGCATGGTGGATGGC 481 ATGATGTTGG TTTTTGGCAA AGGGATTTTG AGTTGCCAGC TCCTCCAAGGCCAGTTAGGC 541 CAGTTACCCA GATCTGAGTC GACCTGCA Sequence 8: NOS terminator(Pst I, -NOS-, Bgl II)   1 GATCGTTCAA ACATTTGGCA ATAAAGTTTC TTAAGATTGAATCCTGTTGC CGGTCTTGCG  61 ATGATTATCA TATAATTTCT GTTGAATTAC GTTAAGCATGTAATAATTAA CATGTAATGC 121 ATGACGTTAT TTATGAGATG GGTTTTTATG ATTAGAGTCCCGCAATTATA CATTTAATAC 181 GCGATAGAAA ACAAAATATA GCGCGCAACC TAGGATAAATTATCGCGCGC GGTGTCATCT 241 ATGTTACTA Sequence 9: Complete sequence ofpZOl502starting at the EcoRI site immediately upstream of the Bt genecassette. The Bt gene (nucleotides 1022-2869) and the pat gene(nucleotides 4294-4845) are aligned with the amino acid sequence of therespective proteins. The recognition sequences of the NotI sitesflanking the beta-lactamase (amp) gene are underlined.    1GAATTCGAGCTCGTCAGAAGACCAGAGCGCTATTGAGACTTTTCAACAAAGGGTAATATCGGGAAACCTCCTCGGATTCC80   81ATTGCCCAGCTATCTGTCACTTCATCGAAAGGACAGTAGAAAAGGAAGGTGGCTCCTACAAATGCCATCATTGCGATAAA160  161GGAAAGGCTATCGTTCAAGATGCCTCTACCGACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAACATCGTGGAAAA240  241AGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGATATCTCCACTGACGTAAGGGATGACGCACAATCCC320  321ACTATCCTTCGCAAGACCCTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGGACACGCTGAAATCACCAGTCTCTC400  401TTCTACAAATCTATCTCTCTCTATTTTCTCCATAATAATGTGTGAGTAGTTCCCAGATAAGGGAATAGGGTTCTTATAGG480  481GTTTCGCTCACGTGTTGAGCATATAAGAAACCCCGAGCTCGGTACCCGGGGATCCGGAAGGTGCAAGGATTGCTCGAGCG560  561TCAAGGATCATTGGTGTCGACCTGAACCCCAGCAGATTCGAAGAAGGTACAGTACACACACATGTATATATGTATGATGT640  641ATCCCTTCGATCGAAGGCATGCCTTGGTATAATCACTGAGTAGTCATTTTATTACTTTGTTTTGACAAGTCAGTAGTTCA720  721TCCATTTGTCCCATTTTTTCAGCTTGGAAGTTTGGTTGCACTGGCACTTGGTCTAATAACTGAGTAGTCATTTTATTACG800  801TTGTTTCGACAAGTCAGTAGCTCATCCATCTGTCCCATTTTTTCAGCTAGGAAGTTTGGTTGCACTGGCCTTGGACTAAT880  881AACTGATTAGTCATTTTATTACATTGTTTCGACAAGTCAGTAGCTCATCCATCTGTCCCATTTTTCAGCTAGGAAGTTCG960  961 GTTGCACTGAATTTGTGAACCCAAAAGACCACAACAAGCCGCGGATCCTCTAGAGTCGACCATG GAC AAC AAC 1033    1                                                              M   D   N   N4 1034 CCA AAC ATC AAC GAA TGC ATT CCA TAC AAC TGC TTG AGT AAC CCA GAAGTT GAA GTA CTT 1093    5P   N   I   N   E   C   I   P   Y   N   C   L   S   N   P   E   V   E   V   L24 1094 GGT GGA GAA CGC ATT GAA ACC GGT TAC ACT CCC ATC GAC ATC TCC TTGTCC TTG ACA CAG 1153   25G   G   E   R   I   E   T   G   Y   T   P   I   D   I   S   L   S   L   T   Q44 1214 TTT CTG CTC AGC GAG TTC GTG CCA GGT GCT GGG TTC GTT CTC GGA CTAGTT GAC ATC ATC 1213   45F   L   L   S   E   F   V   P   G   A   G   F   V   L   G   L   V   D   I   I64 1214 TGG GGT ATC TTT GGT CCA TCT CAA TGG GAT GCA TTC CTG GTG CAA ATTGAG CAG TTG ATC 1273   65W   G   I   F   G   P   S   Q   W   D   A   F   L   V   Q   I   E   Q   L   I84 1274 AAC CAG AGG ATC GAA GAG TTC GCC AGG AAC CAG GCC ATC TCT AGG TTGGAA GGA TTG AGC 1333   85N   Q   R   I   E   E   F   A   R   N   Q   A   I   S   R   L   E   G   L   S104 1334 AAT CTC TAC CAA ATC TAT GCA GAG AGC TTC AGA GAG TGG GAA GCC GATCCT ACT AAC CCA 1393  105N   L   Y   Q   I   Y   A   E   S   F   R   E   W   E   A   D   P   T   N   P124 1394 GCT CTC CGC GAG GAA ATG CGT ATT CAA TTC AAC GAC ATG AAC AGC GCCTTG ACC ACA GCT 1453  125A   L   R   E   E   M   R   I   Q   F   N   D   M   N   S   A   L   T   T   A144 1454 ATC CCA TTG TTC GCA GTC CAG AAC TAC CAA GTT CCT CTC TTG TCC GTGTAC GTT CAA GCA 1513  145I   P   L   F   A   V   Q   N   Y   Q   V   P   L   L   S   V   Y   V   Q   A164 1514 GCT AAT CTT CAC CTC AGC GTG CTT CGA GAC GTT AGC GTG TTT GGG CAAAGG TGG GGA TTC 1573  165A   N   L   H   L   S   V   L   R   D   V   S   V   F   G   Q   R   W   G   F184 1574 GAT GCT GCA ACC ATC AAT AGC CGT TAC AAC GAC CTT ACT AGG CTG ATTGGA AAC TAC ACC 1633  185D   A   A   T   I   N   S   R   Y   N   D   L   T   R   L   I   G   N   Y   T204 1634 GAC CAC GCT GTT CGT TGG TAC AAC ACT GGC TTG GAG CGT GTC TGG GGTCCT GAT TCT AGA 1693  205D   H   A   V   R   W   Y   N   T   G   L   E   R   V   W   G   P   D   S   R224 1694 GAT TGG ATT AGA TAC AAC CAG TTC AGG AGA GAA TTG ACC CTC ACA GTTTTG GAC ATT GTG 1753  225D   W   I   R   Y   N   Q   F   R   R   E   L   T   L   T   V   L   D   I   V244 1754 TCT CTC TTC CCG AAC TAT GAC TCC AGA ACC TAC CCT ATC CGT ACA GTGTCC CAA CTT ACC 1813  245S   L   F   P   N   Y   D   S   R   T   Y   P   I   R   T   V   S   Q   L   T264 1814 AGA GAA ATC TAT ACT AAC CCA GTT CTT GAG AAC TTC GAC GGT AGC TTCCGT GGT TCT GCC 1873  265R   E   I   Y   T   N   P   V   L   E   N   F   D   G   S   F   R   G   S   A284 1874 CAA GGT ATC GAA GGC TCC ATC AGG AGC CCA CAC TTG ATG GAC ATC TTGAAC AGC ATA ACT 1933  285Q   G   I   E   G   S   I   R   S   P   H   L   M   D   I   L   N   S   I   T304 1934 ATC TAC ACC GAT GCT CAC AGA GGA GAG TAT TAC TGG TCT GGA CAC CAGATC ATG GCC TCT 1993  305I   Y   T   D   A   H   R   G   E   Y   Y   W   S   G   H   Q   I   M   A   S324 1994 CCA GTT GGA TTC AGC GGG CCC GAG TTT ACC TTT CCT CTC TAT GGA ACTATG GGA AAC GCC 2053  325P   V   G   F   S   G   P   E   F   T   F   P   L   Y   G   T   M   G   N   A344 2054 GCT CCA CAA CAA CGT ATC GTT GCT CAA CTA GGT CAG GGT GTC TAC AGAACC TTG TCT TCC 2113  345A   P   Q   Q   R   I   V   A   Q   L   G   Q   G   V   Y   R   T   L   S   S364 2114 ACC TTG TAC AGA AGA CCC TTC AAT ATC GGT ATC AAC AAC CAG CAA CTTTCC GTT CTT GAC 2173  365T   L   Y   R   R   P   F   N   I   G   I   N   N   Q   Q   L   S   V   L   D384 2174 GGA ACA GAG TTC GCC TAT GGA ACC TCT TCT AAC TTG CCA TCC GCT GTTTAC AGA AAG AGC 2233  385G   T   E   F   A   Y   G   T   S   S   N   L   P   S   A   V   Y   R   K   S404 2234 GGA ACC GTT GAT TCC TTG GAC GAA ATC CCA CCA CAG AAC AAC AAT GTGCCA CCC AGG CAA 2293  405G   T   V   D   S   L   D   E   I   P   P   Q   N   N   N   V   P   P   R   Q424 2294 GGA TTC TCC CAC AGG TTG AGC CAC GTG TCC ATG TTC CGT TCC GGA TTCAGC AAC AGT TCC 2353  425G   F   S   H   R   L   S   H   V   S   M   F   R   S   G   F   S   N   S   S444 2354 GTG AGC ATC ATC AGA GCT CCT ATG TTC TCA TGG ATT CAT CGT AGT GCTGAG TTC AAC AAT 2413  445V   S   I   I   R   A   P   M   F   S   W   I   H   R   S   A   E   F   N   N464 2414 ATC ATT CCT TCC TCT CAA ATC ACC CAA ATC CCA TTG ACC AAG TCT ACTAAC CTT GGA TCT 2473  465I   I   P   S   S   Q   I   T   Q   I   P   L   T   K   S   T   N   L   G   S484 2474 GGA ACT TCT GTC GTG AAA GGA CCA GGC TTC ACA GGA GGT GAT ATT CTTAGA AGA ACT TCT 2533  485G   T   S   V   V   K   G   P   G   F   T   G   G   D   I   L   R   R   T   S504 2534 CCT GGC CAG ATT AGC ACC CTC AGA GTT AAC ATC ACT GCA CCA CTT TCTCAA AGA TAT CGT 2593  505P   G   Q   I   S   T   L   R   V   N   I   T   A   P   L   S   Q   R   Y   R524 2594 GTC AGG ATT CGT TAC GCA TCT ACC ACA AAC TTG CAA TTC CAC ACC TCCATC GAC GGA AGG 2653  525V   R   I   R   Y   A   S   T   T   N   L   Q   F   H   T   S   I   D   G   R544 2654 CCT ATC AAT CAG GGT AAC TTC TCC GCA ACC ATG TCA AGC GGC AGC AACTTG CAA TCC GGC 2713  545P   I   N   Q   G   N   F   S   A   T   M   S   S   G   S   N   L   Q   S   G564 2714 AGC TTC AGA ACC GTC GGT TTC ACT ACT CCT TTC AAC TTC TCT AAC GGATCA AGC GTT TTC 2773  565S   F   R   T   V   G   F   T   T   P   F   N   F   S   N   G   S   S   V   F584 2774 ACC CTT AGC GCT CAT GTG TTC AAT TCT GGC AAT GAA GTG TAC ATT GACCGT ATT GAG TTT 2833  585T   L   S   A   H   V   F   N   S   G   N   E   V   Y   I   D   R   I   E   F604 2834 GTG CCT GCC GAA GTT ACC TTC GAG GCT GAG TAC TAGCAGATCAGGATCGTTCAAACATTTGGCAATAA 2901  605V   P   A   E   V   T   F   E   A   E   Y   * 616 2902AGTTTCTTAAGATTGAATCCTGTTGCCGGTCTTGCGATGATTATCATATAATTTCTGTTGAATTACGTTAAGCATGTAAT2981 2982AATTAACATGTAATGCATGACGTTATTTATGAGATGGGTTTTTATGATTAGAGTCCCGCAATTATACATTTAATACGCGA3061 3062TAGAAAACAAAATATAGCGCGCAACCTAGGATAAATTATCGCGCGCGGTGTCATCTATGTTACTAGATCCAAGCTTGGCA3141 3142CTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTT3221 3222CGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCC3301 3302TGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTGCTCTGAT3381 3382GCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGC3461 3462TTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAA3541 3542AGGGCCAGATCCGAACATGGTGGAGCACGACACGCTTGTCTACTCCAAAAATATCAAAGATACAGTCTCAGAAGACCAAA3621 3622GGGCAATTGAGACTTTTCAACAAAGGGTAATATCCGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTTATT3701 3702GTGAAGATAGTGGAAAAGGAAGGTGGCTCCTACAAATGCCATCATTGCGATAAAGGAAAGGCCATCGTTGAAGATGCCTC3781 3782TGCCGACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAA3861 3862AGCAAGTGGATTGATGTGATATCTCCACTGACGTAAGGGATGACGCACAATCCCACTATCCTTCGCAAGACCCTTCCTCT3941 3942ATATAAGGAAGTTCATTTCATTTGGAGAGGACACGCTGAAATCACCAGTCTCTCTCTACAAATCTATCTCTCTCTATAAT4021 4022AATGTGTGAGTAGTTCCCAGATAAGGGAATTAGGGTTCTTATAGGGTTTCGCTCATGTGTTGAGCATATAAGAAACCCTT4101 4102ACTCTAGCGAAGATCCTCTTCACCTCGCTCTGCCACACCGACGTCTACTTCTGGGAGGCCAAGGTATCTAATCAGCCATC4181 4182CCATTTGTGATCTTTGTCAGTAGATATGATACAACAACTCGCGGTTGACTTGCGCCTTCTTGGCGGCTTATCTGTCTCAG4261 4262 GGGCAGACTCCCGTGTTCCCTCGGATCTCGAC ATG TCT CCG GAG AGG AGA CCAGTT GAG ATT AGG CCA 4329    1                                 M   S   P   E   R   R   P   V   E   I   R   P12 4330 GCT ACA GCA GCT GAT ATG GCC GCG GTT TGT GAT ATC GTT AAC CAT TACATT GAG ACG TCT 4389   13A   T   A   A   D   M   A   A   V   C   D   I   V   N   H   Y   I   E   T   S32 4390 ACA GTG AAC TTT AGG ACA GAG CCA CAA ACA CCA CAA GAG TGG ATT GATGAT CTA GAG AGG 4449   33T   V   N   F   R   T   E   P   Q   T   P   Q   E   W   I   D   D   L   E   R52 4450 TTG CAA GAT AGA TAC CCT TGG TTG GTT GCT GAG GTT GAG GGT GTT GTGGCT GGT ATT GCT 4509   53L   Q   D   R   Y   P   W   L   V   A   E   V   E   G   V   V   A   G   I   A72 4510 TAC GCT GGG CCC TGG AAG GCT AGG AAC GCT TAC GAT TGG ACA GTT GAGAGT ACT GTT TAC 4569   73Y   A   G   P   W   K   A   R   N   A   Y   D   W   T   V   E   S   T   V   Y92 4570 GTG TCA CAT AGG CAT CAA AGG TTG GGC CTA GGA TCC ACA TTG TAC ACACAT TTG CTT AAG 4629   93V   S   H   R   H   Q   R   L   G   L   G   S   T   L   Y   T   H   L   L   K112 4630 TCT ATG GAG GCG CAA GGT TTT AAG TCT GTG GTT GCT GTT ATA GGC CTTCCA AAC GAT CCA 4689  113S   M   E   A   Q   G   F   K   S   V   V   A   V   I   G   L   P   N   D   P132 4690 TCT GTT AGG TTG CAT GAG GCT TTG GGA TAC ACA GCC CGG GGT ACA TTGCGC GCA GCT GGA 4749  133S   V   R   L   H   E   A   L   G   Y   T   A   R   G   T   L   R   A   A   G152 4750 TAC AAG CAT GGT GGA TGG CAT GAT GTT GGT TTT TGG CAA AGG GAT TTTGAG TTG CCA GCT 4809  153Y   K   H   G   G   W   H   D   V   G   F   W   Q   R   D   F   E   L   P   A172 4810 CCT CCA AGG CCA GTT AGG CCA GTT ACC CAG ATC TGAGTCGACCTGCAGATCGTTCAAACATTTGGCAA 4877  173P   P   R   P   V   R   P   V   T   Q   I   * 184 4878TAAAGTTTCTTAAGATTCAATCCTGTTGCCGGTCTTGCGATGATTATCATATAATTTCTGTTGAATTACGTTAAGCATGT4957 4958AATAATTAACATGTAATGCATGACGTTATTTATGAGATGGGTTTTTATGATTAGAGTCCCGCAATTATACATTTAATACG5037 5038CGATAGAAAACAAAATATAGCGCGCAACCTAGGATAAATTATCGCGCGCGGTGTCATCTATGTTACTAGATCTGGGCCTC5117 5118GTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGT5197 5198GCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGGAGGAGCGGCCGCTCCTCCATG5277 5278AGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTAT5357 5358TCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGT5437 5438TGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTT5517 5518CCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCG5597 5598CCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAA5677 5678GAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAG5757 5758GAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCAT5837 5838ACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTA5917 5918CTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCG5997 5998GCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGG6077 6078TAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGA6157 6158TAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCAT6237 6238TTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGGAGGAGCGGCCGCTCCTCCATGACCAAAATCC6317 6318CTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTG6397 6398CGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCT6477 6478TTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACT6557 6558TCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCG6637 6638TGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACA6717 6718GCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCATTGAGAAAGCGCCACGCTTCCCGAAG6797 6798GGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCC6877 6878TGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAG6957 6958CCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTG7037 7038CGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAG7117 7118CGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTA7197 7198ATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATT7277 7278AGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGA7357 7358 AACAGCTATGACCATGATTAC 7378

SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF SEQUENCES: 11(2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 532 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii)HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (vii) IMMEDIATE SOURCE: (B) CLONE:35S Promoter (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: AATTCGAGCTCGTCAGAAGA CCAGAGGGCT ATTGAGACTT TTCAACAAAG GG TAATATCG 60 GGAAACCTCCTCGGATTCCA TTGCCCAGCT ATCTGTCACT TCATCGAAAG GA CAGTAGAA 120 AAGGAAGGTGGCTCCTACAA ATGCCATCAT TGCGATAAAG GAAAGGCTAT CG TTCAAGAT 180 GCCTCTACCGACAGTGGTCC CAAAGATGGA CCCCCACCCA CGAGGAACAT CG TGGAAAAA 240 GAAGACGTTCCAACCACGTC TTCAAAGCAA GTGGATTGAT GTGATATCTC CA CTGACGTA 300 AGGGATGACGCACAATCCCA CTATCCTTCG CAAGACCCTT CCTCTATATA AG GAAGTTCA 360 TTTCATTTGGAGAGGACACG CTGAAATCAC CAGTCTCTCT CTACAAATCT AT CTCTCTCT 420 ATTTTCTCCATAATAATGTG TGAGTAGTTC CCAGATAAGG GAATTAGGGT AT CTCTCTCT 480 TTTCGCTCACGTGTTGAGCA TATAAGAAAC CCTTACGAGC TCGGTACCCG GG 532 (2) INFORMATION FORSEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 490 base pairs(B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii)ANTI-SENSE: NO (vii) IMMEDIATE SOURCE: (B) CLONE: Adh1-1S intr on 6 (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 2: GATCCGGAAG GTGCAAGGAT TGCTCGAGCGTCAAGGATCA TTGGTGTCGA CC TGAACCCC 60 AGCAGATTCG AAGAAGGTAC AGTACACACACATGTATATA TGTATGATGT AT CCCTTCGA 120 TCGAAGGCAT GCCTTGGTAT AATCACTGAGTAGTCATTTT ATTACTTTGT TT TGACAAGT 180 CAGTAGTTCA TCCATTTGTC CCATTTTTTCAGCTTGGAAG TTTGGTTGCA CT GGCACTTG 240 GTCTAATAAC TGAGTAGTCA TTTTATTACGTTGTTTCGAC AAGTCAGTAG CT CATCCATC 300 TGTCCCATTT TTTCAGCTAG GAAGTTTGGTTGCACTGGCC TTGGACTAAT AA CTGATTAG 360 TCATTTTATT ACATTGTTTC GACAAGTCAGTAGCTCATCC ATCTGTCCCA TT TTTCAGCT 420 AGGAAGTTCG GTTGCACTGA ATTTGTGAACCCAAAAGACC ACAACAAGCC GC GGATCCTC 480 TAGAGTCGAC 490 (2) INFORMATION FORSEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1851 base pairs(B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii)ANTI-SENSE: NO (vii) IMMEDIATE SOURCE: (B) CLONE: cry1Ab toxic generegion (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: CATGGACAAC AACCCAAACATCAACGAATG CATTCCATAC AACTGCTTGA GT AACCCAGA 60 AGTTGAAGTA CTTGGTGGAGAACGCATTGA AACCGGTTAC ACTCCCATCG AC ATCTCCTT 120 GTCCTTGACA CAGTTTCTGCTCAGCGAGTT CGTGCCAGGT GCTGGGTTCG TT CTCGGACT 180 AGTTGACATC ATCTGGGGTATCTTTGGTCC ATCTCAATGG GATGCATTCC TG GTGCAAAT 240 TGAGCAGTTG ATCAACCAGAGGATCGAAGA GTTCGCCAGG AACCAGGCCA TC TCTAGGTT 300 GGAAGGATTG AGCAATCTCTACCAAATCTA TGCAGAGAGC TTCAGAGAGT GG GAAGCCGA 360 TCCTACTAAC CCAGCTCTCCGCGAGGAAAT GCGTATTCAA TTCAACGACA TG AACAGCGC 420 CTTGACCACA GCTATCCCATTGTTCGCAGT CCAGAACTAC CAAGTTCCTC TC TTGTCCGT 480 GTACGTTCAA GCAGCTAATCTTCACCTCAG CGTGCTTCGA GACGTTAGCG TG TTTGGGCA 540 AAGGTGGGGA TTCGATGCTGCAACCATCAA TAGCCGTTAC AACGACCTTA CT AGGCTGAT 600 TGGAAACTAC ACCGACCACGCTGTTCGTTG GTACAACACT GGCTTGGAGC GT GTCTGGGG 660 TCCTGATTCT AGAGATTGGATTAGATACAA CCAGTTCAGG AGAGAATTGA CC CTCACAGT 720 TTTGGACATT GTGTCTCTCTTCCCGAACTA TGACTCCAGA ACCTACCCTA TC CGTACAGT 780 GTCCCAACTT ACCAGAGAAATCTATACTAA CCCAGTTCTT GAGAACTTCG AC GGTAGCTT 840 CCGTGGTTCT GCCCAAGGTATCGAAGGCTC CATCAGGAGC CCACACTTGA TG GACATCTT 900 GAACAGCATA ACTATCTACACCGATGCTCA CAGAGGAGAG TATTACTGGT CT GGACACCA 960 GATCATGGCC TCTCCAGTTGGATTCAGCGG GCCCGAGTTT ACCTTTCCTC TC TATGGAAC 1020 TATGGGAAAC GCCGCTCCACAACAACGTAT CGTTGCTCAA CTAGGTCAGG GT GTCTACAG 1080 AACCTTGTCT TCCACCTTGTACAGAAGACC CTTCAATATC GGTATCAACA AC CAGCAACT 1140 TTCCGTTCTT GACGGAACAGAGTTCGCCTA TGGAACCTCT TCTAACTTGC CA TCCGCTGT 1200 TTACAGAAAG AGCGGAACCGTTGATTCCTT GGACGAAATC CCACCACAGA AC AACAATGT 1260 GCCACCCAGG CAAGGATTCTCCCACAGGTT GAGCCACGTG TCCATGTTCA GT TCCGGATT 1320 CAGCAACAGT TCCGTGAGCATCATCAGAGC TCCTATGTTC TCATGGATTC AT CGTAGTGC 1380 TGAGTTCAAC AATATCATTCCTTCCTCTCA AATCACCCAA ATCCCATTGA CC AAGTCTAC 1440 TAACCTTGGA TCTGGAACTTCTGTCGTGAA AGGACCAGGC TTCACAGGAG GT GATATTCT 1500 TAGAAGAACT TCTCCTGGCCAGATTAGCAC CCTCAGAGTT AACATCACTG CA CCACTTTC 1560 TCAAAGATAT CGTGTCAGGATTCGTTACGC ATCTACCACA AACTTGCAAT TC CACACCTC 1620 CATCGACGGA AGGCCTATCAATCAGGGTAA CTTCTCCGCA ACCATGTCAA GC GGCAGCAA 1680 CTTGCAATCC GGCAGCTTCAGAACCGTCGG TTTCACTACT CCTTTCAACT TC TCTAACGG 1740 ATCAAGCGTT TTCACCCTTAGCGCTCATGT GTTCAATTCT GGCAATGAAG TG TACATTGA 1800 CCGTATTGAG TTTGTGCCTGCCGAAGTTAC CTTCGAGGCT GAGTACTAGC A 1851 (2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 261 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (vii)IMMEDIATE SOURCE: (B) CLONE: NOS terminat or (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 4: GATCAGGATC GTTCAAACAT TTGGCAATAA AGTTTCTTAA GATTGAATCC TGTTGCCGGT 60 CTTGCGATGA TTATCATATA ATTTCTGTTG AATTACGTTA AGCATGTAAT AATTAACATG 120 TAATGCATGA CGTTATTTAT GAGATGGGTT TTTATGATTA GAGTCCCGCA ATTATACATT 180 TAATACGCGA TAGAAAACAA AATATAGCGC GCAACCTAGG ATAAATTATC GCGCGCGGTG 240 TCATCTATGT TACTAGATCC A 261 (2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 560 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (vii)IMMEDIATE SOURCE: (B) CLONE: 35S Promoter (xi) SEQUENCE DESCRIPTION: SEQID NO: 5: GATCCGAACA TGGTGGAGCA CGACACGCTT GTCTACTCCA AAAATATCAA AGATACAGTC 60 TCAGAAGACC AAAGGGCAAT TGAGACTTTT CAACAAAGGG TAATATCCGG AAACCTCCTC 120 GGATTCCATT GCCCAGCTAT CTGTCACTTT ATTGTGAAGA TAGTGGAAAA GGAAGGTGGC 180 TCCTACAAAT GCCATCATTG CGATAAAGGA AAGGCCATCG TTGAAGATGC CTCTGCCGAC 240 AGTGGTCCCA AAGATGGACC CCCACCCACG AGGAGCATCG TGGAAAAAGA AGACGTTCCA 300 ACCACGTCTT CAAAGCAAGT GGATTGATGT GATATCTCCA CTGACGTAAG GGATGACGCA 360 CAATCCCACT ATCCTTCGCA AGACCCTTCC TCTATATAAG GAAGTTCATT TCATTTGGAG 420 AGGACACGCT GAAATCACCA GTCTCTCTCT ACAAATCTAT CTCTCTCTAT AATAATGTGT 480 GAGTAGTTCC CAGATAAGGG AATTAGGGTT CTTATAGGGT TTCGCTCATG TGTTGAGCAT 540 ATAAGAAACC CTTACTCTAG 560 (2) INFORMATION FOR SEQ ID NO: 6:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 180 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (vii)IMMEDIATE SOURCE: (B) CLONE: Adh1-1S intr on 2 (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 6: CGAAGATCCT CTTCACCTCG CTCTGCCACA CCGACGTCTACTTCTGGGAG GC CAAGGTAT 60 CTAATCAGCC ATCCCATTTG TGATCTTTGT CAGTAGATATGATACAACAA CT CGCGGTTG 120 ACTTGCGCCT TCTTGGCGGC TTATCTGTCT CAGGGGCAGACTCCCGTGTT CC CTCGGATC 180 (2) INFORMATION FOR SEQ ID NO: 7: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 568 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE:DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (vii)IMMEDIATE SOURCE: (B) CLONE: Pat gene (xi) SEQUENCE DESCRIPTION: SEQ IDNO: 7: TCGACATGTC TCCGGAGAGG AGACCAGTTG AGATTAGGCC AGCTACAGCA GCTGATATGG 60 CCGCGGTTTG TGATATCGTT AACCATTACA TTGAGACGTC TACAGTGAAC TTTAGGACAG 120 AGCCACAAAC ACCACAAGAG TGGATTGATG ATCTAGAGAG GTTGCAAGAT AGATACCCTT 180 GGTTGGTTGC TGAGGTTGAG GGTGTTGTGG CTGGTATTGC TTACGCTGGG CCCTGGAAGG 240 CTAGGAACGC TTACGATTGG ACAGTTGAGA GTACTGTTTA CGTGTCACAT AGGCATCAAA 300 GGTTGGGCCT AGGATCCACA TTGTACACAC ATTTGCTTAA GTCTATGGAG GCGCAAGGTT 360 TTAAGTCTGT GGTTGCTGTT ATAGGCCTTC CAAACGATCC ATCTGTTAGG TTGCATGAGG 420 CTTTGGGATA CACAGCCCGG GGTACATTGC GCGCAGCTGG ATACAAGCAT GGTGGATGGC 480 ATGATGTTGG TTTTTGGCAA AGGGATTTTG AGTTGCCAGC TCCTCCAAGG CCAGTTAGGC 540 CAGTTACCCA GATCTGAGTC GACCTGCA 568 (2) INFORMATION FOR SEQID NO: 8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 249 base pairs (B)TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii)MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO(vii) IMMEDIATE SOURCE: (B) CLONE: NOS Terminat or (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 8: GATCGTTCAA ACATTTGGCA ATAAAGTTTC TTAAGATTGAATCCTGTTGC CG GTCTTGCG 60 ATGATTATCA TATAATTTCT GTTGAATTAC GTTAAGCATGTAATAATTAA CA TGTAATGC 120 ATGACGTTAT TTATGAGATG GGTTTTTATG ATTAGAGTCCCGCAATTATA CA TTTAATAC 180 GCGATAGAAA ACAAAATATA GCGCGCAACC TAGGATAAATTATCGCGCGC GG TGTCATCT 240 ATGTTACTA 249 (2) INFORMATION FOR SEQ ID NO:9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 7378 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (vii)IMMEDIATE SOURCE: (B) CLONE: Complete seq uence of pZO1502 (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 9: GAATTCGAGC TCGTCAGAAG ACCAGAGGGC TATTGAGACTTTTCAACAAA GG GTAATATC 60 GGGAAACCTC CTCGGATTCC ATTGCCCAGC TATCTGTCACTTCATCGAAA GG ACAGTAGA 120 AAAGGAAGGT GGCTCCTACA AATGCCATCA TTGCGATAAAGGAAAGGCTA TC GTTCAAGA 180 TGCCTCTACC GACAGTGGTC CCAAAGATGG ACCCCCACCCACGAGGAACA TC GTGGAAAA 240 AGAAGACGTT CCAACCACGT CTTCAAAGCA AGTGGATTGATGTGATATCT CC ACTGACGT 300 AAGGGATGAC GCACAATCCC ACTATCCTTC GCAAGACCCTTCCTCTATAT AA GGAAGTTC 360 ATTTCATTTG GAGAGGACAC GCTGAAATCA CCAGTCTCTCTCTACAAATC TA TCTCTCTC 420 TATTTTCTCC ATAATAATGT GTGAGTAGTT CCCAGATAAGGGAATTAGGG TT CTTATAGG 480 GTTTCGCTCA CGTGTTGAGC ATATAAGAAA CCCCGAGCTCGGTACCCGGG GA TCCGGAAG 540 GTGCAAGGAT TGCTCGAGCG TCAAGGATCA TTGGTGTCGACCTGAACCCC AG CAGATTCG 600 AAGAAGGTAC AGTACACACA CATGTATATA TGTATGATGTATCCCTTCGA TC GAAGGCAT 660 GCCTTGGTAT AATCACTGAG TAGTCATTTT ATTACTTTGTTTTGACAAGT CA GTAGTTCA 720 TCCATTTGTC CCATTTTTTC AGCTTGGAAG TTTGGTTGCACTGGCACTTG GT CTAATAAC 780 TGAGTAGTCA TTTTATTACG TTGTTTCGAC AAGTCAGTAGCTCATCCATC TG TCCCATTT 840 TTTCAGCTAG GAAGTTTGGT TGCACTGGCC TTGGACTAATAACTGATTAG TC ATTTTATT 900 ACATTGTTTC GACAAGTCAG TAGCTCATCC ATCTGTCCCATTTTTCAGCT AG GAAGTTCG 960 GTTGCACTGA ATTTGTGAAC CCAAAAGACC ACAACAAGCCGCGGATCCTC TA GAGTCGAC 1020 CATGGACAAC AACCCAAACA TCAACGAATG CATTCCATACAACTGCTTGA GT AACCCAGA 1080 AGTTGAAGTA CTTGGTGGAG AACGCATTGA AACCGGTTACACTCCCATCG AC ATCTCCTT 1140 GTCCTTGACA CAGTTTCTGC TCAGCGAGTT CGTGCCAGGTGCTGGGTTCG TT CTCGGACT 1200 AGTTGACATC ATCTGGGGTA TCTTTGGTCC ATCTCAATGGGATGCATTCC TG GTGCAAAT 1260 TGAGCAGTTG ATCAACCAGA GGATCGAAGA GTTCGCCAGGAACCAGGCCA TC TCTAGGTT 1320 GGAAGGATTG AGCAATCTCT ACCAAATCTA TGCAGAGAGCTTCAGAGAGT GG GAAGCCGA 1380 TCCTACTAAC CCAGCTCTCC GCGAGGAAAT GCGTATTCAATTCAACGACA TG AACAGCGC 1440 CTTGACCACA GCTATCCCAT TGTTCGCAGT CCAGAACTACCAAGTTCCTC TC TTGTCCGT 1500 GTACGTTCAA GCAGCTAATC TTCACCTCAG CGTGCTTCGAGACGTTAGCG TG TTTGGGCA 1560 AAGGTGGGGA TTCGATGCTG CAACCATCAA TAGCCGTTACAACGACCTTA CT AGGCTGAT 1620 TGGAAACTAC ACCGACCACG CTGTTCGTTG GTACAACACTGGCTTGGAGC GT GTCTGGGG 1680 TCCTGATTCT AGAGATTGGA TTAGATACAA CCAGTTCAGGAGAGAATTGA CC CTCACAGT 1740 TTTGGACATT GTGTCTCTCT TCCCGAACTA TGACTCCAGAACCTACCCTA TC CGTACAGT 1800 GTCCCAACTT ACCAGAGAAA TCTATACTAA CCCAGTTCTTGAGAACTTCG AC GGTAGCTT 1860 CCGTGGTTCT GCCCAAGGTA TCGAAGGCTC CATCAGGAGCCCACACTTGA TG GACATCTT 1920 GAACAGCATA ACTATCTACA CCGATGCTCA CAGAGGAGAGTATTACTGGT CT GGACACCA 1980 GATCATGGCC TCTCCAGTTG GATTCAGCGG GCCCGAGTTTACCTTTCCTC TC TATGGAAC 2040 TATGGGAAAC GCCGCTCCAC AACAACGTAT CGTTGCTCAACTAGGTCAGG GT GTCTACAG 2100 AACCTTGTCT TCCACCTTGT ACAGAAGACC CTTCAATATCGGTATCAACA AC CAGCAACT 2160 TTCCGTTCTT GACGGAACAG AGTTCGCCTA TGGAACCTCTTCTAACTTGC CA TCCGCTGT 2220 TTACAGAAAG AGCGGAACCG TTGATTCCTT GGACGAAATCCCACCACAGA AC AACAATGT 2280 GCCACCCAGG CAAGGATTCT CCCACAGGTT GAGCCACGTGTCCATGTTCC GT TCCGGATT 2340 CAGCAACAGT TCCGTGAGCA TCATCAGAGC TCCTATGTTCTCATGGATTC AT CGTAGTGC 2400 TGAGTTCAAC AATATCATTC CTTCCTCTCA AATCACCCAAATCCCATTGA CC AAGTCTAC 2460 TAACCTTGGA TCTGGAACTT CTGTCGTGAA AGGACCAGGCTTCACAGGAG GT GATATTCT 2520 TAGAAGAACT TCTCCTGGCC AGATTAGCAC CCTCAGAGTTAACATCACTG CA CCACTTTC 2580 TCAAAGATAT CGTGTCAGGA TTCGTTACGC ATCTACCACAAACTTGCAAT TC CACACCTC 2640 CATCGACGGA AGGCCTATCA ATCAGGGTAA CTTCTCCGCAACCATGTCAA GC GGCAGCAA 2700 CTTGCAATCC GGCAGCTTCA GAACCGTCGG TTTCACTACTCCTTTCAACT TC TCTAACGG 2760 ATCAAGCGTT TTCACCCTTA GCGCTCATGT GTTCAATTCTGGCAATGAAG TG TACATTGA 2820 CCGTATTGAG TTTGTGCCTG CCGAAGTTAC CTTCGAGGCTGAGTACTAGC AG ATCAGGAT 2880 CGTTCAAACA TTTGGCAATA AAGTTTCTTA AGATTGAATCCTGTTGCCGG TC TTGCGATG 2940 ATTATCATAT AATTTCTGTT GAATTACGTT AAGCATGTAATAATTAACAT GT AATGCATG 3000 ACGTTATTTA TGAGATGGGT TTTTATGATT AGAGTCCCGCAATTATACAT TT AATACGCG 3060 ATAGAAAACA AAATATAGCG CGCAACCTAG GATAAATTATCGCGCGCGGT GT CATCTATG 3120 TTACTAGATC CAAGCTTGGC ACTGGCCGTC GTTTTACAACGTCGTGACTG GG AAAACCCT 3180 GGCGTTACCC AACTTAATCG CCTTGCAGCA CATCCCCCTTTCGCCAGCTG GC GTAATAGC 3240 GAAGAGGCCC GCACCGATCG CCCTTCCCAA CAGTTGCGCAGCCTGAATGG CG AATGGCGC 3300 CTGATGCGGT ATTTTCTCCT TACGCATCTG TGCGGTATTTCACACCGCAT AT GGTGCACT 3360 CTCAGTACAA TCTGCTCTGA TGCCGCATAG TTAAGCCAGCCCCGACACCC GC CAACACCC 3420 GCTGACGCGC CCTGACGGGC TTGTCTGCTC CCGGCATCCGCTTACAGACA AG CTGTGACC 3480 GTCTCCGGGA GCTGCATGTG TCAGAGGTTT TCACCGTCATCACCGAAACG CG CGAGACGA 3540 AAGGGCCAGA TCCGAACATG GTGGAGCACG ACACGCTTGTCTACTCCAAA AA TATCAAAG 3600 ATACAGTCTC AGAAGACCAA AGGGCAATTG AGACTTTTCAACAAAGGGTA AT ATCCGGAA 3660 ACCTCCTCGG ATTCCATTGC CCAGCTATCT GTCACTTTATTGTGAAGATA GT GGAAAAGG 3720 AAGGTGGCTC CTACAAATGC CATCATTGCG ATAAAGGAAAGGCCATCGTT GA AGATGCCT 3780 CTGCCGACAG TGGTCCCAAA GATGGACCCC CACCCACGAGGAGCATCGTG GA AAAAGAAG 3840 ACGTTCCAAC CACGTCTTCA AAGCAAGTGG ATTGATGTGATATCTCCACT GA CGTAAGGG 3900 ATGACGCACA ATCCCACTAT CCTTCGCAAG ACCCTTCCTCTATATAAGGA AG TTCATTTC 3960 ATTTGGAGAG GACACGCTGA AATCACCAGT CTCTCTCTACAAATCTATCT CT CTCTATAA 4020 TAATGTGTGA GTAGTTCCCA GATAAGGGAA TTAGGGTTCTTATAGGGTTT CG CTCATGTG 4080 TTGAGCATAT AAGAAACCCT TACTCTAGCG AAGATCCTCTTCACCTCGCT CT GCCACACC 4140 GACGTCTACT TCTGGGAGGC CAAGGTATCT AATCAGCCATCCCATTTGTG AT CTTTGTCA 4200 GTAGATATGA TACAACAACT CGCGGTTGAC TTGCGCCTTCTTGGCGGCTT AT CTGTCTCA 4260 GGGGCAGACT CCCGTGTTCC CTCGGATCTC GACATGTCTCCGGAGAGGAG AC CAGTTGAG 4320 ATTAGGCCAG CTACAGCAGC TGATATGGCC GCGGTTTGTGATATCGTTAA CC ATTACATT 4380 GAGACGTCTA CAGTGAACTT TAGGACAGAG CCACAAACACCACAAGAGTG GA TTGATGAT 4440 CTAGAGAGGT TGCAAGATAG ATACCCTTGG TTGGTTGCTGAGGTTGAGGG TG TTGTGGCT 4500 GGTATTGCTT ACGCTGGGCC CTGGAAGGCT AGGAACGCTTACGATTGGAC AG TTGAGAGT 4560 ACTGTTTACG TGTCACATAG GCATCAAAGG TTGGGCCTAGGATCCACATT GT ACACACAT 4620 TTGCTTAAGT CTATGGAGGC GCAAGGTTTT AAGTCTGTGGTTGCTGTTAT AG GCCTTCCA 4680 AACGATCCAT CTGTTAGGTT GCATGAGGCT TTGGGATACACAGCCCGGGG TA CATTGCGC 4740 GCAGCTGGAT ACAAGCATGG TGGATGGCAT GATGTTGGTTTTTGGCAAAG GG ATTTTGAG 4800 TTGCCAGCTC CTCCAAGGCC AGTTAGGCCA GTTACCCAGATCTGAGTCGA CC TGCAGATC 4860 GTTCAAACAT TTGGCAATAA AGTTTCTTAA GATTGAATCCTGTTGCCGGT CT TGCGATGA 4920 TTATCATATA ATTTCTGTTG AATTACGTTA AGCATGTAATAATTAACATG TA ATGCATGA 4980 CGTTATTTAT GAGATGGGTT TTTATGATTA GAGTCCCGCAATTATACATT TA ATACGCGA 5040 TAGAAAACAA AATATAGCGC GCAACCTAGG ATAAATTATCGCGCGCGGTG TC ATCTATGT 5100 TACTAGATCT GGGCCTCGTG ATACGCCTAT TTTTATAGGTTAATGTCATG AT AATAATGG 5160 TTTCTTAGAC GTCAGGTGGC ACTTTTCGGG GAAATGTGCGCGGAACCCCT AT TTGTTTAT 5220 TTTTCTAAAT ACATTCAAAT ATGTATCCGC TCATGGAGGAGCGGCCGCTC CT CCATGAGA 5280 CAATAACCCT GATAAATGCT TCAATAATAT TGAAAAAGGAAGAGTATGAG TA TTCAACAT 5340 TTCCGTGTCG CCCTTATTCC CTTTTTTGCG GCATTTTGCCTTCCTGTTTT TG CTCACCCA 5400 GAAACGCTGG TGAAAGTAAA AGATGCTGAA GATCAGTTGGGTGCACGAGT GG GTTACATC 5460 GAACTGGATC TCAACAGCGG TAAGATCCTT GAGAGTTTTCGCCCCGAAGA AC GTTTTCCA 5520 ATGATGAGCA CTTTTAAAGT TCTGCTATGT GGCGCGGTATTATCCCGTAT TG ACGCCGGG 5580 CAAGAGCAAC TCGGTCGCCG CATACACTAT TCTCAGAATGACTTGGTTGA GT ACTCACCA 5640 GTCACAGAAA AGCATCTTAC GGATGGCATG ACAGTAAGAGAATTATGCAG TG CTGCCATA 5700 ACCATGAGTG ATAACACTGC GGCCAACTTA CTTCTGACAACGATCGGAGG AC CGAAGGAG 5760 CTAACCGCTT TTTTGCACAA CATGGGGGAT CATGTAACTCGCCTTGATCG TT GGGAACCG 5820 GAGCTGAATG AAGCCATACC AAACGACGAG CGTGACACCACGATGCCTGT AG CAATGGCA 5880 ACAACGTTGC GCAAACTATT AACTGGCGAA CTACTTACTCTAGCTTCCCG GC AACAATTA 5940 ATAGACTGGA TGGAGGCGGA TAAAGTTGCA GGACCACTTCTGCGCTCGGC CC TTCCGGCT 6000 GGCTGGTTTA TTGCTGATAA ATCTGGAGCC GGTGAGCGTGGGTCTCGCGG TA TCATTGCA 6060 GCACTGGGGC CAGATGGTAA GCCCTCCCGT ATCGTAGTTATCTACACGAC GG GGAGTCAG 6120 GCAACTATGG ATGAACGAAA TAGACAGATC GCTGAGATAGGTGCCTCACT GA TTAAGCAT 6180 TGGTAACTGT CAGACCAAGT TTACTCATAT ATACTTTAGATTGATTTAAA AC TTCATTTT 6240 TAATTTAAAA GGATCTAGGT GAAGATCCTT TTTGATAATCTCATGGAGGA GC GGCCGCTC 6300 CTCCATGACC AAAATCCCTT AACGTGAGTT TTCGTTCCACTGAGCGTCAG AC CCCGTAGA 6360 AAAGATCAAA GGATCTTCTT GAGATCCTTT TTTTCTGCGCGTAATCTGCT GC TTGCAAAC 6420 AAAAAAACCA CCGCTACCAG CGGTGGTTTG TTTGCCGGATCAAGAGCTAC CA ACTCTTTT 6480 TCCGAAGGTA ACTGGCTTCA GCAGAGCGCA GATACCAAATACTGTCCTTC TA GTGTAGCC 6540 GTAGTTAGGC CACCACTTCA AGAACTCTGT AGCACCGCCTACATACCTCG CT CTGCTAAT 6600 CCTGTTACCA GTGGCTGCTG CCAGTGGCGA TAAGTCGTGTCTTACCGGGT TG GACTCAAG 6660 ACGATAGTTA CCGGATAAGG CGCAGCGGTC GGGCTGAACGGGGGGTTCGT GC ACACAGCC 6720 CAGCTTGGAG CGAACGACCT ACACCGAACT GAGATACCTACAGCGTGAGC AT TGAGAAAG 6780 CGCCACGCTT CCCGAAGGGA GAAAGGCGGA CAGGTATCCGGTAAGCGGCA GG GTCGGAAC 6840 AGGAGAGCGC ACGAGGGAGC TTCCAGGGGG AAACGCCTGGTATCTTTATA GT CCTGTCGG 6900 GTTTCGCCAC CTCTGACTTG AGCGTCGATT TTTGTGATGCTCGTCAGGGG GG CGGAGCCT 6960 ATGGAAAAAC GCCAGCAACG CGGCCTTTTT ACGGTTCCTGGCCTTTTGCT GG CCTTTTGC 7020 TCACATGTTC TTTCCTGCGT TATCCCCTGA TTCTGTGGATAACCGTATTA CC GCCTTTGA 7080 GTGAGCTGAT ACCGCTCGCC GCAGCCGAAC GACCGAGCGCAGCGAGTCAG TG AGCGAGGA 7140 AGCGGAAGAG CGCCCAATAC GCAAACCGCC TCTCCCCGCGCGTTGGCCGA TT CATTAATG 7200 CAGCTGGCAC GACAGGTTTC CCGACTGGAA AGCGGGCAGTGAGCGCAACG CA ATTAATGT 7260 GAGTTAGCTC ACTCATTAGG CACCCCAGGC TTTACACTTTATGCTTCCGG CT CGTATGTT 7320 GTGTGGAATT GTGAGCGGAT AACAATTTCA CACAGGAAACAGCTATGACC AT GATTAC 7378 (2) INFORMATION FOR SEQ ID NO: 10: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 615 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: unknown (D) TOPOLOGY: linear (ii) MOLECULE TYPE:protein (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (vi) ORIGINALSOURCE: (A) ORGANISM: Bacillus thuringiensis (vii) IMMEDIATE SOURCE: (B)CLONE: Bt protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: Met Asp AsnAsn Pro Asn Ile Asn Glu Cys Il e Pro Tyr Asn Cys Leu 1 5 10 15 Ser AsnPro Glu Val Glu Val Leu Gly Gly Gl u Arg Ile Glu Thr Gly 20 25 30 TyrThr Pro Ile Asp Ile Ser Leu Ser Leu Th r Gln Phe Leu Leu Ser 35 40 45Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gl y Leu Val Asp Ile Ile 50 5560 Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Al a Phe Leu Val Gln Ile 6570 75 80 Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Ph e Ala Arg Asn GlnAla 85 90 95 Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Ty r Gln Ile TyrAla Glu 100 105 110 Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr As n Pro AlaLeu Arg Glu 115 120 125 Glu Met Arg Ile Gln Phe Asn Asp Met Asn Se r AlaLeu Thr Thr Ala 130 135 140 Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Va lPro Leu Leu Ser Val 145 1 50 1 55 1 60 Tyr Val Gln Ala Ala Asn Leu HisLeu Ser Va l Leu Arg Asp Val Ser 165 170 175 Val Phe Gly Gln Arg Trp GlyPhe Asp Ala Al a Thr Ile Asn Ser Arg 180 185 190 Tyr Asn Asp Leu Thr ArgLeu Ile Gly Asn Ty r Thr Asp His Ala Val 195 200 205 Arg Trp Tyr Asn ThrGly Leu Glu Arg Val Tr p Gly Pro Asp Ser Arg 210 215 220 Asp Trp Ile ArgTyr Asn Gln Phe Arg Arg Gl u Leu Thr Leu Thr Val 225 2 30 2 35 2 40 LeuAsp Ile Val Ser Leu Phe Pro Asn Tyr As p Ser Arg Thr Tyr Pro 245 250 255Ile Arg Thr Val Ser Gln Leu Thr Arg Glu Il e Tyr Thr Asn Pro Val 260 265270 Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Se r Ala Gln Gly Ile Glu 275280 285 Gly Ser Ile Arg Ser Pro His Leu Met Asp Il e Leu Asn Ser Ile Thr290 295 300 Ile Tyr Thr Asp Ala His Arg Gly Glu Tyr Ty r Trp Ser Gly HisGln 305 3 10 3 15 3 20 Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pr o GluPhe Thr Phe Pro 325 330 335 Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gl nGln Arg Ile Val Ala 340 345 350 Gln Leu Gly Gln Gly Val Tyr Arg Thr LeuSe r Ser Thr Leu Tyr Arg 355 360 365 Arg Pro Phe Asn Ile Gly Ile Asn AsnGln Gl n Leu Ser Val Leu Asp 370 375 380 Gly Thr Glu Phe Ala Tyr Gly ThrSer Ser As n Leu Pro Ser Ala Val 385 3 90 3 95 4 00 Tyr Arg Lys Ser GlyThr Val Asp Ser Leu As p Glu Ile Pro Pro Gln 405 410 415 Asn Asn Asn ValPro Pro Arg Gln Gly Phe Se r His Arg Leu Ser His 420 425 430 Val Ser MetPhe Arg Ser Gly Phe Ser Asn Se r Ser Val Ser Ile Ile 435 440 445 Arg AlaPro Met Phe Ser Trp Ile His Arg Se r Ala Glu Phe Asn Asn 450 455 460 IleIle Pro Ser Ser Gln Ile Thr Gln Ile Pr o Leu Thr Lys Ser Thr 465 4 70 475 4 80 Asn Leu Gly Ser Gly Thr Ser Val Val Lys Gl y Pro Gly Phe Thr Gly485 490 495 Gly Asp Ile Leu Arg Arg Thr Ser Pro Gly Gl n Ile Ser Thr LeuArg 500 505 510 Val Asn Ile Thr Ala Pro Leu Ser Gln Arg Ty r Arg Val ArgIle Arg 515 520 525 Tyr Ala Ser Thr Thr Asn Leu Gln Phe His Th r Ser IleAsp Gly Arg 530 535 540 Pro Ile Asn Gln Gly Asn Phe Ser Ala Thr Me t SerSer Gly Ser Asn 545 5 50 5 55 5 60 Leu Gln Ser Gly Ser Phe Arg Thr ValGly Ph e Thr Thr Pro Phe Asn 565 570 575 Phe Ser Asn Gly Ser Ser Val PheThr Leu Se r Ala His Val Phe Asn 580 585 590 Ser Gly Asn Glu Val Tyr IleAsp Arg Ile Gl u Phe Val Pro Ala Glu 595 600 605 Val Thr Phe Glu Ala GluTyr 610 615 (2) INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 183 amino acids (B) TYPE: amino acid (C)STRANDEDNESS: unknown (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein(iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (vii) IMMEDIATE SOURCE: (B)CLONE: Pat protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: Met Ser ProGlu Arg Arg Pro Val Glu Ile Ar g Pro Ala Thr Ala Ala 1 5 10 15 Asp MetAla Ala Val Cys Asp Ile Val Asn Hi s Tyr Ile Glu Thr Ser 20 25 30 ThrVal Asn Phe Arg Thr Glu Pro Gln Thr Pr o Gln Glu Trp Ile Asp 35 40 45Asp Leu Glu Arg Leu Gln Asp Arg Tyr Pro Tr p Leu Val Ala Glu Val 50 5560 Glu Gly Val Val Ala Gly Ile Ala Tyr Ala Gl y Pro Trp Lys Ala Arg 6570 75 80 Asn Ala Tyr Asp Trp Thr Val Glu Ser Thr Va l Tyr Val Ser HisArg 85 90 95 His Gln Arg Leu Gly Leu Gly Ser Thr Leu Ty r Thr His LeuLeu Lys 100 105 110 Ser Met Glu Ala Gln Gly Phe Lys Ser Val Va l Ala ValIle Gly Leu 115 120 125 Pro Asn Asp Pro Ser Val Arg Leu His Glu Al a LeuGly Tyr Thr Ala 130 135 140 Arg Gly Thr Leu Arg Ala Ala Gly Tyr Lys Hi sGly Gly Trp His Asp 145 1 50 1 55 1 60 Val Gly Phe Trp Gln Arg Asp PheGlu Leu Pr o Ala Pro Pro Arg Pro 165 170 175 Val Arg Pro Val Thr Gln Ile180

What is claimed is:
 1. Seed of maize inbred line R412H having beendeposited under ATCC Accession No. 209675, further comprising a nucleicacid construct comprising two cassettes, wherein the first cassettecomprises a CaMV 35S constitutive promoter operably linked to a maizealcohol dehydrogenase intron, a DNA sequence of a gene encoding a Cry1Abprotein, and a terminator functional in plants, and the second cassettecomprises a CaMV 35S promoter which functions in plant cells operablylinked to a maize alcohol dehydrogenase intron, a DNA sequence of a geneencoding for phosphinothricin acetyl transferase, and a terminatorfunctional in plants, wherein the two cassettes are transcribed in thesame direction, wherein the nucleic acid construct is incorporated intothe seed's genome on chromosome 8, near position 117, between markersZ1B3 and UMC150a.
 2. Seed according to claim 1, wherein the firstexpression cassette comprises SEQ ID Nos. 1-4 in operable sequence. 3.Seed according to claim 1, wherein the second expression cassettecomprises SEQ ID Nos. 5-8 in operable sequence.
 4. Seed according toclaim 1, wherein the first expression cassette comprises SEQ ID Nos. 1-4in operable sequence and the second expression cassette comprises SEQ IDNos. 5-8 in operable sequence.
 5. A maize plant, or parts thereof, ofinbred line R412H, seed of said line having been deposited under ATCCaccession No: 209675, further comprising a nucleic acid constructcomprising two cassettes, wherein the first cassette comprises a CaMV35S constitutive promoter operably linked to a maize alcoholdehydrogenase intron, a DNA sequence of a gene encoding a Cry1Abprotein, and a terminator functional in plants, and the second cassettecomprises a CaMV 35S promoter which functions in plant cells operablylinked to a maize alcohol debydrogenase intron, a DNA sequence of a geneencoding for phosphinothricin acetyl transferase, and a terminatorfunctional in plants, wherein the two cassettes are transcribed in thesame direction, wherein the nucleic acid construct is incorporated intothe seed's genome on chromosome 8, near position 117, between markersZ1B3 and UMC150a.
 6. A maize plant according to claim 5, wherein thefirst expression cassette comprises SEQ ID Nos. 1-4 in operablesequence.
 7. A maize plant according to claim 5, wherein the secondexpression cassette comprises SEQ ID Nos. 5-8 in operable sequence.
 8. Amaize plant according to claim 5, wherein the first expression cassettecomprises SEQ ID Nos. 1-4 in operable sequence and the second expressioncassette comprises SEQ ID Nos. 5-8 in operable sequence.
 9. Pollen ofthe plant of claim
 5. 10. An ovule of the plant of claim
 5. 11. A maizeplant, or parts thereof, having all the genotypic and phenotypiccharacteristics of a plant according to claim
 5. 12. Hybrid maize seedproduced by crossing a plant according to claim 5 with an inbred maizeplant having a different genotype.
 13. Hybrid maize plant produced bygrowing hybrid maize seed of claim
 12. 14. A method of producing hybridmaize seeds comprising the following steps: (a) planting seeds of afirst inbred maize line according to claim 1 and seeds of a secondinbred line having a different genotype; (b) cultivating maize plantsresulting from said planting until time of flowering; (c) emasculatingsaid flowers of plants of one of the maize inbred lines; (d) allowingpollination of the other inbred line to occur, and (e) harvesting thehybrid seeds produced thereby.
 15. Hybrids seed produced by the methodof claim
 14. 16. Hybrid maize plant produced by growing hybrid maizeseed of claim 15.